Antibacterial Compositions

ABSTRACT

The present invention relates to oral antibacterial compositions comprising trihydroxybenzoate derivatives, e.g., useful for the treatment of gum diseases (e.g., gingivitis or periodontitis) and to methods of using such compositions.

RELATED APPLICATIONS

This application is a Divisional Application of U.S. Ser. No.12/762,234, filed on Apr. 16, 2010, which claims the benefit of U.S.Provisional Application No. 61/169,845 filed Apr. 16, 2009, entitled“Antibacterial Compositions” by Toshihisa Kawai, et al.

The entire teachings of the above application are incorporated herein byreference.

BACKGROUND

Gum disease is an example of a bacterial infection that affects a numberof people. Gingivitis and periodontitis are common gum diseases that areproblematic and, in certain instances, can be difficult to prevent ortreat.

A need exists for an oral formulation that has anti-bacterialproperties. In particular, a further need exists for oral formationsthat are effective in the prevention or treatment of gum disease andother bacterial infections.

SUMMARY OF THE INVENTION

The present invention relates to oral antibacterial compositionscomprising trihydroxybenzoate derivatives, e.g., useful for thetreatment of gum diseases (e.g., gingivitis or periodontitis) and tomethods of using such compositions.

Surprisingly, we have found that an important pharmacophore in thesecompounds is trihydroxybenzoate. Compounds having trihydroxybenzoatemoieties are effective primarily not by any direct antimicrobial action,but rather by potently stimulating the production of certain endogenousantimicrobial peptides, particularly LL-37 peptide, from epithelialcells. Trihydroxybenzoates stimulate LL-37 production by binding to theDEAD box polypeptide-S (DDX5).

The present invention relates provide oral care compositions forinhibiting oral bacteria, their uses and methods of treatment thereof,comprising trihydroxybenzoate derivatives. The current inventionprovides a method of stimulating the production of antimicrobialpeptides in mammalian cells comprising contacting said cells with aneffective amount of trihydroxybenzoate derivatives in free or salt form.The present invention further includes assessing a level of stimulationof the production of antimicrobial peptides; wherein an increase in thestimulation the production of antimicrobial peptides occurs, as comparedto a control. Trihydroxybenzoate derivatives useful for the currentinvention include 3,4,5-trihydroxybenzoic acid (i.e., gallic acid), itsisoform, 2,3,4-trihydroxybenzoic acid,C₁₋₄alkyl-3,4,5-trihydroxybenzoate (e.g., ethyl3,4,5-trihydroxybenzoate), C₁₋₄alkyl-2,3,4-trihydroxybenzoate (e.g.,ethyl 2,3,4-trihydroxybenzoate), EGCG and mixtures thereof, in free orsalt form (Trihydroxybenzoate Derivatives of the Invention).

In particular, it is surprisingly found that TrihydroxybenzoateDerivatives of the present invention increases the production ofendogenous antimicrobial peptides, particularly LL-37 in gingival andcheek mucosal epithelial cells, thereby efficiently suppresses bacterial(e.g., Actinobacillus actinomycetemcomitans) growth known to cause gumdiseases. Significantly, trihydroxybenzoate derivatives of the presentinvention induce endogenous antibiotic peptide production withoutinducing the expression of host pro-inflammatory factors such ascytokines (e.g., TNFa, IL-1b and IL-6), eicosanoids (e.g., prostaglandinE2 and Leukotriene B4) that are known to elicit, augment and prolonginflammatory conditions.

Therefore, in a particular embodiment, the present invention provides anoral care composition (Composition 1) useful for inhibiting oralbacteria, comprising a polyphenol extract enriched withtrihydroxybenzoate derivatives selected from one or more3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, C₁₋₄alkyl3,4,5-trihydroxybenzoate (e.g., ethyl 3,4,5-trihydroxybenzoate),C₁₋₄alkyl-2,3,4-trihydroxybenzoate (e.g., ethyl2,3,4-trihydroxybenzoate), 3,4,5-trihydroxybenzoic acid,2,3,4-trihydroxybenzoic acid, EGCG and mixtures thereof in free or saltform, e.g., enriched with at least 0.12 wt. %, preferably 0.12-10 wt. %,preferably 0.15-3 wt. %, more preferably 0.5-1.5 wt. %, most preferably0.6 wt. % of trihydroxybenzoate derivatives. In a preferred embodiment,the extract is enriched with C₁₋₄alkyl 3,4,5-trihydroxybenzoate,C₁₋₄alkyl-2,3,4-trihydroxybenzoate or mixtures thereof. In yet anotherpreferred embodiment, the extract is enriched with 2,3,4-hydroxybenzoicacid, 3,4,5-trihydroxybenzoic acid or mixtures thereof in free or saltform. In yet another preferred embodiment, the extract is enriched withEGCG.

Furthermore, it is also surprisingly found that TrihydroxybenzoateDerivatives of the Invention efficiently stimulate the production ofantimicrobial peptides LL-37 in epithelial cells while dihydroxycatechins such as catechins and epicatechins show little or no inductionof LL-37 expression in epithelial cells. Therefore, in anotherembodiment, the invention provides an oral care composition (Composition2) comprising trihydroxybenzoate derivatives selected from one or more3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, C₁₋₄alkyl3,4,5-trihydroxybenzoate (e.g., ethyl 3,4,5-trihydroxybenzoate),C₁₋₄alkyl-2,3,4-trihydroxybenzoate (e.g., ethyl2,3,4-trihydroxybenzoate), EGCG, catechin gallate and epicatechingallate, and mixtures thereof in free or salt form, wherein saidcomposition is substantially free of catechins having only adihydroxybenzyl moiety, e.g., catechin and epicatechin, e.g., whereinthe ratios of Trihydroxybenzoate Derivatives of the Invention todihydroxy catechins is less than 10%, preferably less than 1%, morepreferably less than 0.1%, more preferably less than 0.05%, even morepreferably less than 0.01%, most preferably less than 0.001% of theamount of dihyroxy catechins generally found in tea extracts. In onepreferred embodiment, Composition 2 comprises trihydroxybenzoatederivatives selected from 3,4,5-trihydroxybenzoic acid,2,3,4-trihydroxybenzoic acid and mixtures thereof in free or salt form,wherein said composition is substantially free of catechin andepicatechin (other than catechin gallate and epicatechin gallate, whichcontain trihydroxybenzyl moiety). In another preferred embodiment,Composition 2 comprises trihydroxybenzoate derivatives selected from2,3,4-trihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid and mixturesthereof in free or salt form, wherein said composition is substantiallyfree of catechin and. In yet another preferred embodiment, Composition 2comprises EGCG, wherein said composition is substantially free ofcatechin and epicatechins

In yet another embodiment, the invention further provides an oral carecomposition (Composition 3) comprising trihydroxybenzoate derivativesselected from selected from 3,4,5-trihydroxybenzoic acid,2,3,4-trihydroxybenzoic acid, C₁₋₄alkyl 3,4,5-trihydroxybenzoate (e.g.,ethyl 3,4,5-trihydroxybenzoate), C₁₋₄alkyl 2,3,4-trihydroxybenzoate(e.g., ethyl 2,3,4-trihydroxybenzoate), EGCG or mixtures thereof in freeor salt form, wherein said composition is substantially free ofdihydroxy catechins and GCG, e.g., wherein the amounts of dihydroxycatechins and GCG is less than 10%, preferably less than 1%, morepreferably less than 0.1%, more preferably less than 0.05%, even morepreferably less than 0.01%, most preferably less than 0.001% of theamount of dihyroxy catechins and GCG generally found in tea extracts. Ina specific embodiment, said dihydroxy catechins are catechins andepicatechins In yet another specific embodiment, the trihydroxybenzoatederivatives are selected from 3,4,5-trihydroxybenzoic acid,2,3,4-trihydroxybenzoic acid and mixture thereof in free or salt form.In yet another specific embodiment, the trihydroxybenzoate derivativesare selected from C₁₋₄alkyl-3,4,5-trihydroxybenzoate (e.g.,ethyl-3,4,5-trihydroxybenzoate), C₁₋₄alkyl-2,3,4-trihydroxybenzoate(e.g., ethyl 2,3,4-trihydroxybenzoate) and mixtures thereof. In yetanother specific embodiment, the trihydroxybenzoate derivative is EGCG.

In still yet another embodiment, the invention provides an oral carecomposition (Composition 4) comprising trihydroxybenzoate derivativesselected from 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoicacid, C₁₋₄alkyl 3,4,5-trihydroxybenzoate (e.g., ethyl3,4,5-trihydroxybenzoate), C₁₋₄alkyl-2,3,4-trihydroxybenzoate (e.g.,ethyl 2,3,4-trihydroxybenzoate), EGCG and mixtures thereof in free orsalt form, wherein said composition is substantially free of otherpolyphenols (e.g., p-coumaric acid, caffeic acid, vanillic acid, ferulicacid, p-hydroxybenzoic acid, ellagic acid, anthocyanins, catechins,flavan-3-ols catechin, epicatechin, gallocatechin, epigallocatechin,epicatechin 3-O-gallate, catechin gallate, epicatechin gallate,quercetin, kaempferol, myricetin, fisetin, isoquercitrin, hyperoside,genistein, daidzei, lignins, proanthocyanidins, resveratrol andtannins), e.g., wherein these other polyphenols comprise less than 5 wt.% of the total composition, preferably less than 1%, more preferablyless than 0.5 wt. %, even more preferably less than 0.05 wt. % and mostpreferably less than 0.01 wt. % of the total weight of the composition.In an embodiment, the composition of the present invention includesabout 0.0001% to about 2% by weight of a Camellia extract, said extractcomprises trihydroxybenzoate derivatives in a concentration from about0.1 ug/ml to about 100 ug/ml; wherein the composition stimulate LL-37production in gingival cells. The composition can further includes0.001% to about 5% by weight of fluoride ion sources, water, abrasives,surfactants, foaming agents, vitamins, polymers, enzymes, humectants,thickeners, antimicrobial agents, preservatives, flavorings, coloringsand a combinations thereof.

In yet another specific embodiment, the trihydroxybenzoate derivativesare selected from 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoicacid and mixture thereof in free or salt form. In yet another specificembodiment, the trihydroxybenzoate derivatives are selected fromC₁₋₄alkyl-3,4,5-trihydroxybenzoate (e.g.,ethyl-3,4,5-trihydroxybenzoate), C₁₋₄alkyl 2,3,4-trihydroxybenzoate(e.g., ethyl 2,3,4-trihydroxybenzoate) and mixtures thereof. In yetanother specific embodiment, the trihydroxybenzoate derivative is EGCG.

In a particular embodiment the invention provides a composition(Composition 5), comprises trihydroxybenzoate compounds, e.g., asdescribed above for any of Compositions 1-4, wherein thetrihydroxybenzoate compounds are shown to

-   -   (i) Bind to or have an affinity for DDX5 and/or    -   (ii) Stimulate LL-37 production.        For example, the invention provides a plant extract comprising        trihydroxybenzoate compounds which extract is concentrated with        compounds binding to DDX5, e.g., obtained using affinity        purification, e.g., affinity chromatography or beads or other        surface coated with DDX5 and/or a fragment or derivative of DDX5        capable of binding a trihydroxybenzoate compound. The invention        likewise provides methods of making plant extracts comprising        passing a solution over a surface coated with DDX5 and/or a        fragment or derivative of DDX5 capable of binding a        trihydroxybenzoate compound.

The oral composition may be in the form of a solid or semi-solid such astoothpaste, chewing gum or lozenges; or in a liquid such as a mouthrinse.

In another embodiment, the invention further provides Composition 1, 2,3, 4 or 5, further comprises one or more of a fluoride ion source,water, abrasives, surfactants, foaming agents, vitamins, polymers,enzymes, humectants, thickeners, antimicrobial agents, preservatives,flavorings, colorings and/or combinations thereof.

In a particular embodiment, the invention provides Composition 1, 2, 3,4 or 5 as follows:

-   -   5.1. Any of Composition 1-5, further comprising an abrasive.    -   5.2. Any of Composition 1-5, or 5.1, wherein the abrasive is        selected from a calcium phosphate (e.g., dicalcium phosphate        dihydrate), calcium sulfate, precipitated calcium carbonate,        silica (e.g. hydrated silica), and combinations thereof.    -   5.3. Any of the preceding compositions comprising an abrasive in        an amount of about 15 wt. % to about 70 wt. % of the total        composition weight.    -   5.4. Any of the preceding compositions comprising a small        particle abrasive having a d50 of <5 micrometers.    -   5.5. Any of the preceding compositions comprising at least one        fluoride ion source.    -   5.6. Formula 5.5, wherein said fluoride ion source is selected        from sodium fluoride, potassium fluoride and ammonium fluoride.    -   5.7. Formula 5.5 or 5.6, wherein said fluoride ion source        provides from about 50-10,000 ppm, preferably, 100-1000 ppm,        most preferably about 500 ppm.    -   5.8. Formula 5.5, 5.6 or 5.7, wherein said fluoride ion source        is in the amount of about 0.15 wt. % to about 2.5 wt. % of the        total composition weight.    -   5.9. Any of the preceding compositions comprising at least one        surfactant.    -   5.10. Any of the preceding compositions comprising at least one        surfactant selected from sodium lauryl sulfate, cocamidopropyl        betaine, and combinations thereof.    -   5.11. Any of the preceding compositions comprising an anionic        surfactant.    -   5.12. Any of the preceding compositions comprising sodium lauryl        sulfate.    -   5.13. Any of the preceding compositions comprising at least one        humectant.    -   5.14. Any of the preceding compositions comprising at least one        humectant selected from glycerin, sorbitol and combinations        thereof    -   5.15. Any of the preceding compositions comprising at least one        polymer.    -   5.16. Any of the preceding compositions comprising at least one        polymer selected from polyethylene glycols, polyvinylmethyl        ether maleic acid copolymers, polysaccharides (e.g. cellulose        derivatives, for example carboxymethyl cellulose, or        polysaccharide gums, for example xanthan gum or carrageenan        gum), and combinations thereof.    -   5.17. Any of the preceding compositions comprising gum strips or        fragments.    -   5.18. Any of the preceding compositions comprising flavoring,        fragrance and/or coloring.    -   5.19. Any of the preceding compositions comprising water.    -   5.20. Any of the preceding compositions comprising an        antibacterial agent.    -   5.21. Any of the preceding compositions comprising an        antibacterial agent selected from triclosan, herbal extracts and        essential oils (e.g. rosemary extract, thymol, menthol,        eucalyptol, methyl salicylate), bisguanide antiseptics (e.g.,        chlorhexidine, alexidine or octenidine), quaternary ammonium        compounds (e.g., cetylpyridinium chloride), phenolic        antiseptics, hexetidine, povidone iodine, delmopinol, salifluor,        metal ions (e.g., zinc salts, for example, zinc citrate),        sanguinarine, propolis and oxygenating agents (e.g., hydrogen        peroxide, buffered sodium peroxyborate or peroxycarbonate).    -   5.22. Any of the preceding compositions comprising triclosan.    -   5.23. Any of the preceding compositions comprising an        antibacterial agent in an amount of 0.01-5 wt. % of the total        composition weight.    -   5.24. Any of the preceding compositions comprising triclosan in        an amount of 0.01 to 1 wt. percent of the total composition        weight.    -   5.25. Any of the preceding compositions comprising triclosan in        an amount of about 0.3% of the total composition weight.    -   5.26. Any of the preceding compositions effective upon        application to the oral cavity, e.g., with brushing, to improve        oral health by:        -   (i) reducing or inhibiting oral bacteria;        -   (ii) preventing or treating gum diseases, oral malodor,            and/or tooth decay;        -   (iii) reducing or inhibiting pre-carious lesions of the            enamel;        -   (iv) reducing or inhibiting demineralization of the teeth;        -   (v) reducing hypersensitivity of the teeth;        -   (vi) reducing or inhibiting formation of dental caries;        -   (vii) promoting healing of sores or cuts in the mouth;        -   (viii) reducing levels of acid producing bacteria;        -   (ix) increasing relative levels of antimicrobial LL-37            peptides in the oral cavity; and/or        -   (x) reducing plaque accumulation.    -   5.27. A composition obtained or obtainable by combining the        ingredients as set forth in any of the preceding compositions.    -   5.28. Any of the preceding compositions wherein the composition        is in the form of toothpaste.    -   5.29. Any of the preceding compositions wherein the composition        is a toothpaste optionally further comprising one or more of one        or more of water, abrasives, surfactants, foaming agents,        vitamins, polymers, enzymes, humectants, thickeners,        antimicrobial agents, preservatives, flavorings, colorings        and/or combinations thereof.    -   5.30. Any of Composition 1-5 or 5.1-5.27, wherein the        composition is in the form of a mouthwash.    -   5.31. Any of Composition 1-5 or 5.1-5.27, wherein the        composition is chewing gum.    -   5.32. Any of Composition 1-5 or 5.1-5.27, wherein said        composition is in the form of a gel dentifrice.

As the invention uses a composition enriched with trihydroxybenzoatederivatives that is effective in stimulating LL-37 peptides, thecompositions of the invention can optionally comprise other polyphenols,but in a smaller amount than normally would required in conventionaloral care compositions for improving oral hygiene.

The invention thus further encompasses methods to improve oral health(Method 1) by (i) reducing or inhibiting oral bacteria, (ii) preventingor treating gum diseases (e.g., gingivitis or periodontitis) oralmalodor, and/or tooth decay, (iii) reducing or inhibiting pre-cariouslesions of the enamel, e.g., as detected by quantitative light-inducedfluorescence (QLF) or electrical conductance measurement (ECM), (iv)reducing or inhibiting demineralization of the teeth, (v) reducinghypersensitivity of the teeth, (vi) reducing or inhibiting formation ofdental caries, (vii) promoting healing of sores or cuts in the mouth,(viii) reducing levels of acid producing bacteria, (ix) increasingrelative levels of antimicrobial LL-37 peptides, and/or (x) reducingplaque accumulation, comprising applying a Composition of the Invention,as described herein, e.g., Compositions 1-5 or any of 5.1-5.32, to theoral cavity, e.g., by applying a Composition of the Invention to theoral cavity of a mammal in need thereof.

Accordingly, the present invention pertains to methods of treating anindividual having an oral cavity disease or condition by selecting anindividual having an oral cavity disease or condition, wherein the oralcavity disease or condition involves a decreased level of antimicrobialpeptide stimulation; and administering an amount of trihydroxybenzoatederivatives to the oral cavity; wherein the stimulation of theproduction of antimicrobial peptides occurs. Examples of oral cavitydiseases or conditions includes thrush, gingivitis, periodontitis, oralcandidiasis, salivary diseases associated with bacterial, viral, orfungal infections; Crohn's disease, irritable bowel syndrome, ulcerativecolitis, Helicobacter pylori caused stomach ulcer, and gastrointestinaldiseases associated with mucosal bacteria. The steps of the presentinvention allow for the stimulation of the production of antimicrobialpeptide reduces one or more symptoms associated with the oral cavitydisease or condition.

Similarly, the method of the present invention includes stimulating theproduction of antimicrobial peptides in cells of an individual, byselecting an individual having a decreased level of antimicrobialpeptide stimulation, as compared to a level of antimicrobial peptidestimulation of a control; contacting said cells with an effective amountof one or more trihydroxybenzoate derivatives; wherein an increase inthe stimulation of the production of antimicrobial peptides occurs, ascompared to the decreased level of antimicrobial peptide stimulationprior to contacting the cells. The level of antimicrobial peptidestimulation can be assessed prior to contact the cells or after, orboth. In an embodiment, the method includes assessing the level of LL-37production.

It is known that dental plaque is a biofilm consists primarily ofbacteria (e.g., streptococcus mutans and anaerobes) which deposits atthe teeth. Plaque is the main etiological factor responsible for dentalcaries and periodontal diseases such as gingivitis, periodontitis, oralmalodor, and/or tooth decay. By reducing or inhibiting oral bacteria inthe dental cavity through the stimulation of the production ofantimicrobial peptides such as LL-37 and hBD2 in gingival epithelialcells using the Compositions of the Invention, said compositions iseffective in producing the conditions provided above in Method 1.Similarly, dental hypersensitivity and demineralization of teeth resultwhen protective enamel is lost due to the acid by-product of thebacterial degradation of fermentable carbohydrate, these conditions maybe ameliorated by increasing production of antimicrobial peptides in thegingival epithelial cells. Likewise, by increasing antimicrobialpeptides, particularly peptide LL-3 in the gingival, thereby reducingpathogenic bacteria, the Compositions of the Invention are useful forpromote healing of sores or cuts in the mouth.

The present invention includes methods for treating a bacterialinfection in an individual by administering an amount of any compositiondescribed herein to the individual, wherein the bacterial infection isreduced. Examples of such bacterial infection include thrush,gingivitis, periodontitis, oral candidiasis, salivary diseasesassociated with bacterial, viral, or fungal infections; Crohn's disease,irritable bowel syndrome, ulcerative colitis, Helicobacter pylori causedstomach ulcer, and gastrointestinal diseases associated with mucosalbacteria.

In addition, the present invention is also useful for treating otherbacterial conditions such as keratitis, conjunctivitis,keratoconjunctivitis and corneal blindness. Keratitis is a bacterial(e.g., staphylococcus aureus) infection of the cornea generally due toinjury or from wearing contact lenses. Ocular LL-37 peptides possesspotent antibacterial activity against Gram-Positive and Gram-negativebacteria most often responsible for bacterial keratitis as well asagainst the most common Gram-positive keratitis-producing organisms SEand SA. Moreover, LL-37 possess significant antiviral inhibitoryactivity against herpes simplex virus (HSV-1), which is the leadingcause of corneal blindness. Similarly, LL-37 has demonstratedsignificant inhibitory activity in vitro against Ad 19, which is themajor cause of conjunctivitis and epidemic keratoconjunctivitis.Therefore, by stimulating the production of LL-37, the Compositions ofthe Invention is useful for keratitis, conjunctivitis,keratoconjunctivitis and corneal blindness.

As mentioned above, the present invention provides a composition for andmethod of stimulating production of anti-microbial peptide LL-37 inepithelial cells without inducing pro-inflammatory factors such ascytokines (e.g., TNFa, IL-1b and IL-7) and eicosanoids (e.g.,Prostagladin E2 and Leukotriene B4). As such, the Compositions of theinvention are particularly useful for treating a bacterial infectionwherein (a) the bacteria is antibiotic resistant, (b) the patient isallergic to antibiotics; (c) the patient is immuno-compromised (e.g.,HIVIAIDS patients); or (d) the infection is of an oral cavity. Patientswho are likely to benefit from the current invention include, forexample, HIV/AIDS patients experiencing thrush.

Additionally, LL-37 is a potent antimicrobial against variousstaphylococcal species. However, the antibiotics currently available forstaphylococcal infections are derived from synthetic compounds such thatthey can generate more virulent antibiotic-resistant strains ofpathogenic bacteria, such as those found in Methicilin-resistantStaphylococcus aureus (MRSA). As trihydroxybenzoate derivatives of thecurrent invention work by increasing the host's intrinsic peptideantibiotic production, such compositions may be used to treat MRSA,particularly in those patients who are immune-compromised as in HIV/AIDSpatients or patients who have recently undergone transplant or arehypersensitive to traditional antibiotics used to treat theseinfections.

In another embodiment, the invention further provides a gene switchcomprising a heterologous gene under control of an LL-37 promoter,stimulated by trihydroxybenzoate derivatives selected from3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, c1-4-alkyl3,4,5-trihydroxybenzoate (e.g., ethyl 3,4,5-trihydroxybenzoate),C₁₋₄alkyl 2,3,4-trihydroxybenzoate (e.g., ethyl2,3,4-trihydroxybenzoate), EGCG, GCG and mixtures thereof in free orsalt form.

In yet another embodiment, the present invention provides a screeningmethod for measuring LL-37 expression, using trihydroxybenzoatederivatives as candidate or as positive control. In a furtherembodiment, said trihydroxybenzoate derivatives are selected from one ormore 3,4,5-trihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid,C₁₋₄alkyl 3,4,5-trihydroxybenzoate (e.g., ethyl3,4,5-trihydroxybenzoate), C₁₋₄alkyl 2,3,4-trihydroxybenzoate (e.g.,ethyl-2,3,4-trihydroxybenzoate), EGCG, GCG and mixtures thereof in freeor salt form.

In a further example, the present invention provides a method ofscreening for compounds useful as antimicrobial and anti-inflammatorycompounds, e.g., by stimulating LL-37 expression, comprising measuringthe binding of test compounds to DDX5. The present invention involvesmethods of obtaining a plant extract enriched for molecules that bind toDDX5 comprising contacting the material to be extracted to a surfacecoated with DDX5 or a fragment of derivative of DDX5 that binds atrihydroxybenzoate derivative.

Another embodiment of the present invention includes methods forscreening for one or more compounds that stimulate the production of oneor more antibacterial peptides by contacting the compound to be testedwith the DDX5 polypeptide sufficiently to allow formation of a complexbetween the compound to be tested and the isolated polypeptide, tothereby form a complex; and assessing the presence, absence or amount ofthe complex; wherein the presence of the complex indicates that thecompound stimulates production of one or more antibacterial peptides;and the absence of a complex indicates that the compound does notstimulate the production of one or more antibacterial peptides. Themethod can include, e.g., comparing the amount of the complex with to acontrol (e.g., wherein the control is the amount of LL-37-antibodycomplex obtained under the same conditions). The isolated polypeptide orcompound to be tested can be detectably labeled. The method furtherincludes, in an embodiment, contacting the complex with an antibodyspecific to said DDX5 or said complex. DDX5 or the antibody can be boundto a solid support.

The present invention relates to methods for assessing a compound forstimulation of an anti-microbial peptide in a sample by contacting thesample with a compound to be tested to allow stimulation of theanti-microbial peptide; contacting the sample with an antibody thatbinds to the anti-microbial peptide sufficient to allow formation of acomplex between the sample and the antibody, to thereby form anantigen-antibody complex; and assessing the presence, absence or amountof the antigen-antibody complex; wherein the presence or an increasedlevel of the anti-microbial peptide, as compared to a control, indicatesthat the compound stimulates production of the anti-microbial peptide,and the absence or decreased level of anti-microbial peptide, ascompared to a control, indicates that the compound does not stimulate ofthe production of the anti-microbial peptide. Examples of antimicrobialpeptides include LL-37, hBD2, hBD3, Ghrelin, Lysozyme or combinationthereof. The method can also include comparing the amount of theantigen-antibody complex to a control. The antibody can be detectablylabeled. The method further includes contacting the sample with a secondantibody specific to the antigen or antigen-antibody complex. Theanti-microbial peptide or the antibody can be bound to a solid support.In an embodiment, a trihydroxybenzoate derivative is used as a positivecontrol.

The present invention further relates to methods for assessing acompound for stimulation of an anti-microbial peptide in a sample bycontacting the sample with the compound to be tested sufficiently toallow stimulation of anti-microbial peptide; contacting the sample withat least two oligonucleotide primers in a polymerase chain reaction,wherein at least one of the oligonucleotide primers is specific for thenucleic acid sequence of anti-microbial peptide, sufficiently to allowamplification of the primers; and detecting in the sample the amplifiednucleic acid sequence; wherein the presence the amplified nucleic acidsequence indicates that the compound stimulates production of theanti-microbial peptide, and the absence of the amplified nucleic acidsequence indicates that the compound does not stimulate of theproduction of the anti-microbial peptide. At least one of theoligonucleotide primers can include at least about 10 contiguous bases.

The present invention further embodies methods for assessing a compoundfor stimulation of an anti-microbial peptide in a sample by contactingthe sample with the compound to be tested sufficiently to allowstimulation of the anti-microbial peptide; contacting the sample withone or more oligonucleotide probes specific for an isolated nucleic acidmolecule of the anti-microbial peptide under high stringency conditions,sufficiently to allow hybridization between the sample and the probe;and detecting the nucleic acid molecule that hybridizes to theoligonucleotide probe in the sample; wherein the presence thehybridization indicates that the compound stimulates production of theanti-microbial peptide, and the absence of hybridization indicates thatthe compound does not stimulate of the production of the anti-microbialpeptide

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing a pull down assay to determine counterligand of benzoic acids expressed on OBA9 cells is DDX5. OBA9 cells wereharvested and dissolved in lysis buffer. The resulting cell lysissolution was reacted with Sephadex beads conjugated with 2,6-dihydroxybenzoic acid (DHBC) and 2,3,4-trihydroxy benzoic acid (THBC). Thecellular components pulled down with beads were separated in SDS-PAGEgel, and stained with Coomassie blue. The 35 kD and 70 kD bands specificto 2,3,4-THBC were subjected to Mass-Spec analysis at Tufts UniversityCore Facility. The procedure for pull down assay includes preparation ofcell lysate from gingival epithelial cell (OBA9) culture; preparation ofbeads conjugated with tri-hydroxy benzoic acid (THBC); incubation ofbeads with OBA9 cell lysate; remove unbound protein by washing beads;the proteins bound to beads are separated in SDS-PAGE; distinct bands to2,3,4-THBC are sent for Mass-Spec service; and identification ofmolecular finger print of 2,3,4-THBC binding ligand.

FIG. 2 is a schematic including the molecular finger print found in 70kD band specific to 2,3,4-THDC that is DDX5 (SEQ ID NO: 1). The 35 kD,70 kD and 90 kD bands specific to 2,3,4-THBC were subjected to Mass-Specanalysis service. While 35 kD and 90 kD bands did not contain anydistinct protein(s) to 2,3,4-THDC beads, 70 kD band contain DEAD boxpolypeptide 5 (DDX5), which was only found in 2,3,4-THDC beads. Resultdata sheet that identified DDX5 in 70 kD band is shown.

FIG. 3 is a photograph of blots showing suppression of DDX5 (by RT-PCR:DDX5 mRNA, β-actin mRNA and by western blot: DDX5 protein using SEQ IDNOs: 2-7).

FIG. 4 is a photograph of a western blot showing the effects of DDX5suppression on EGCG-mediated LL-37 mRNA expression in OBA9 cells (lanes:1 and 3; Control no treatment 2 and 4; Control none target siRNA; 5:DDX5 siRNA sequence A; 6; DDX5 siRNA sequence B).

FIG. 5 is a graph showing the effects of DDX5 suppression onEGCG-mediating LL-37 expression (in ng/ml) in OBA9 cells over time.

FIG. 6 is a photograph of blots showing over expression of DDX5 (byRT-PCR: DDX5 mRNA, β-actin mRNA and by western blot: DDX5 protein).

FIG. 7 is a bar graph showing the effects of DDX5 over expression onEGCG-mediated LL-37 expression (in ng/ml) in OBA9 cells.

DETAILED DESCRIPTION OF THE INVENTION

“THBC” refers to chemical structures such as trihydroxybenzoates thatcomprise tri-hydroxy benzoic acid and its isoforms, which are found inthe polyphenol extracts of natural plant products or in antioxidant foodpreservatives. THBC has been found to bind DEAD box polypeptide 5 (DDX5)expressed in the gingival epithelial cells. It was determined that 1)THBC present in epigallocatechin gallate (EGCG) binds to DDX5 present inthe cytoplasm of gingival epithelial cells and 2) suppression of DDX5expression in gingival epithelial cells using RNAi-specific to DDX5 mRNAresulted in inhibition of both mRNA and protein expression of LL-37 inthe EGCG-stimulated gingival epithelial cells.

These findings prove that THBC acts directly on the molecule present inthe cytoplasm of gingival epithelial cells. The finding that DDX5 is aligand of EGCG in normal (non-cancer) cells demonstrates a novelbiological mechanism elicited by EGCG.

THBC is thus useful for 1) oral health care products, 2) skin andmucosal health care products, 3) alternatives to, or supplements for,antimicrobial drugs, and 4) neutraceutical products.

The finding of EGCG-binding to DDX5 provides additional potential usesfor THBC, including 1) DDX5 as a drug discovery target molecule and 2)detection of DDX5 in human and animal tissue samples as a diagnosticindicator of host innate immune potency.

In a further example, the invention provides a method of screening forcompounds useful as antimicrobial and anti-inflammatory compounds, e.g.,by stimulating LL-37 expression, comprising measuring the binding oftest compounds to DDX5.

Using the anti-LL-37 antibodies, compounds that stimulate LL-37 and/orare mediated by DDX5 via the LL-37 pathway can be assessed using theassays described herein.

Most currently available oral and body health care products for treatingand preventing inflammation rely on either direct action to host cellsby anti-inflammatory drugs or on antibiotic action againstmicroorganisms. THBC, however, utilizes a completely novel biologicalpathway. Specifically, THBC acts on host cells to increase the hostbody's production of antimicrobial peptides through its binding to DDX5.No agent is currently available that acts in a like manner to increasethe host body's natural power to fight off microorganisms or to bindDDX5.

Moreover, most antibiotics currently used to prevent or treat infectionsare derived from synthetic compounds. As such, they can generate muchmore virulent antibiotic-resistant strains of pathogenic bacteria. Forexample, the emergence of antibiotic-resistant bacteria, such asMethicillin-resistant Staphylococcus aureus (MRSA), is one of thesignificant issues in medicine today. However, THBC, as noted above, isnot a synthetic antibiotic. It actually increases the host body'sintrinsic antibiotic peptide production. Thus, THBC is expected to be animportant alternative where a diagnosis requires that an anti-infectionregimen be antibiotic-free.

Compounds that can specifically bind DDX5 are considered to act in amanner similar to THBC, and are for example useful to an increase LL-37expression in host cells. The signal-transduction molecules which conveysignals elicited by THBC-activated DDX5 should also be involved in theup-regulation of LL-37 expression in host cells. In this case, theelucidation of such signal-transduction molecules downstream of DDX5 mayestablish another targets for drug discovery.

The biological action of THBC-binding of DDX5 to elicit LL-37 expressionin host cells provides additional data about innate immunity, which isuseful to investigators involved in basic medical and pharmacologicalresearch. Specifically, the cytoplasmic molecules currently known toelicit innate immune responses are nucleotide-binding oligomerizationdomain NOD and several other molecules. However, no studies have everreported DDX5 as an innate immune response molecule. Therefore, thediscovery of DDX5, as an innate immune regulatory molecule, enhances ourunderstanding of the innate immune system and how to developtechnologies dedicated to its manipulation.

THBC includes trihydroxybenzoate derivatives. As used herein, the term“trihydroxybenzoate derivatives” include 3,4,5-trihydroxybenzoic acid,2,3,4-trihydroxybenzoic acid, C₁₋₄alkyl 3,4,5-trihydroxybenzoate (e.g.,ethyl 3,4,5-trihydroxybenzoate), C₁₋₄alkyl 2,3,4-trihydroxybenzoate(e.g., ethyl 2,3,4-trihydroxybenzoate), EGCG, and mixtures thereof infree or salt form. In particular embodiments, for example, in methodsfor stimulating production of antimicrobial peptides, for treatingkeratitis, conjunctivitis, keratoconjunctivitis and corneal blindness,and for measuring LL-37 expression, the term trihydroxybenzoatederivatives further include GCG.

The term “dihydroxy catechins” herein refers to catchin and epicatechin,but does not include catechin galate and epicatechin gallate.

The term “polyphenols” herein refers to a large family of compoundscommonly found in a wide variety of plants, fruits and vegetables.Polyphenols include, but not limited to (1) phenolic acid (e.g.,p-coumaric, caffeic, vanillic, ferulic, p-hydroxybenzoic, gallic andellagic acid) esters or glycosides conjugated with other naturalcompounds such as flavonoids, alcohols, hydroxyfatty acids, sterols, andglucosides; and (2) flavanoids which comprise of anthocyanins,catechins, flavan-3-ols catechin, epicatechin, gallocatechin,epigallocatechin, epicatechin 3-O-gallate, catechin gallate, epicatechingallate, quercetin, kaempferol, myricetin, fisetin, isoquercitrin,hyperoside, genistein, daidzei, lignins, proanthocyanidins, resveratroland tannins)

The phrase “substantially free of” as used herein refers to compositionswherein the ratio of Trihydroxybenzoate Derivatives of the Invention to,for example, dihydroxy catechins (e.g., catechin or epicatechin or GCGis less than 10%, preferably less than 1%, more preferably less than0.1%, more preferably less than 0.05%, even more preferably less than0.01%, most preferably less than 0.001% of the amount of dihyroxycatechins generally found in tea extracts, particularly green teaextract such as Oolong tea and green tea. It has been found thatdifferent kinds of tea extracts generally contain the following amountsof flavanols:

Flavonols Amounts (g) per 100 g of dry mass EGCG 0.93-5.74 EGC 0.73-3.25ECG 0.17-1.16 C 0.04-0.48 EC 0.1-0.8 Gallic acid 0.06-0.62 Theogallin0.17-0.97 Caffeine 2.25-4.33

Catechin and epicatechin therefore generally make up about 0.1-1.3% oftea extracts. Accordingly, a useful composition of the inventioncomprises trihydroxybenzoate derivatives and an amount of tea extractwherein said extract contains less than 0.14% of catechins andepicatechins. In a specific embodiment wherein the composition issubstantially free of dihydroxybenzoate catechins and GCG, thecomposition contains, for example, less than 10%, preferably less than1%, more preferably less than 0.1%, more preferably less than 0.05%,even more preferably less than 0.01%, most preferably less than 0.001%of the amount of dihyroxy catechins and GCG generally found in teaextracts. Wherein the composition is substantially free of otherpolyphenols (i.e., substantially free of polyphenols except for EGCG andgallic acid), said composition comprises trihydroxybenzoate derivativesand optionally polyphenols in an amount less than 5 wt. % of the totalcomposition, preferably less than 1%, more preferably less than 0.5 wt.%, even more preferably less than 0.05 wt. % and most preferably lessthan 0.01 wt. % of the total weight of the composition.

The phrase “enriched with trihydroxybenzoate derivatives” herein refersto a composition wherein the total amounts of trihydroxybenzoatederivatives are at least 200% of the amount of gallic acid in polyphenolextracts, e.g., at least 0.12 wt. % of the total tea extract, preferably0.12-10.00 wt. %, preferably 0.15-3.00 wt. %, more preferably 0.5-1.5wt. %, most preferably 0.6 wt. %.

Fluoride Ion Source

Fluoride ion sources are well known for use in oral compositions asanti-caries agents and are preferably, but not necessarily contained inthe Compositions of the Invention to provide the oral health benefits ofthe invention. Fluoride ions are contained in a number of oral carecompositions for this purpose, particularly toothpastes. Patentsdisclosing such toothpastes include U.S. Pat. No. 3,538,230, Nov. 3,1970 to Pader et al; U.S. Pat. No. 3,689,637, Sep. 5, 1972 to Pader;U.S. Pat. No. 3,711,604, Jan. 16, 1973 to Colodney et al; U.S. Pat. No.3,911,104, Oct. 7, 1975 to Harrison; U.S. Pat. No. 3,935,306, Jan. 27,1976 to Roberts et al; and U.S. Pat. No. 4,040,858, Aug. 9, 1977 toWason, the contents of all of which are herein incorporated byreference.

Addition of fluoride ions to the Compositions of the Invention serves toprotect teeth against decay. A wide variety of fluoride ion-yieldingmaterials can be employed as sources of soluble fluoride in the instantcompositions. Examples of suitable fluoride ion-yielding materials arefound in Briner et al; U.S. Pat. No. 3,535,421; issued Oct. 20, 1970 andWidder et al; U.S. Pat. No. 3,678,154; issued Jul. 18, 1972, thecontents of which are hereby incorporated by reference. Preferredfluoride ion sources for use herein include sodium fluoride, potassiumfluoride and ammonium fluoride. Sodium fluoride is particularlypreferred. Preferably the instant compositions provide from about50-10,000 ppm, preferably, 100-1000 ppm, most preferably about 500 ppmof fluoride ions in the compositions that contact dental surfaces whenused with the compositions of the present invention. Generally, thefluoride will be present at a level of from about 0.15% to about 2.5% byweight of the composition.

Abrasives

The Compositions of the Invention may include one or more abrasives, forexample silica abrasives such as precipitated silicas having a meanparticle size of up to about 20 microns, such as Zeodent 115®, marketedby J. M. Huber. Other useful abrasives also include sodiummetaphosphate, potassium metaphosphate, aluminum silicate, calcinedalumina, bentonite or other siliceous materials, or combinationsthereof.

The silica abrasive polishing materials useful herein, as well as theother abrasives, generally have an average particle size ranging betweenabout 0.1 and 30 microns, about between 5 and about 15 microns. Thesilica abrasives can be from precipitated silica or silica gels, such asthe silica xerogels described in U.S. Pat. No. 3,538,230, to Pader etal. and U.S. Pat. No. 3,862,307, to Digiulio, both incorporated hereinby reference. Particular silica xerogels are marketed under the tradename Syloid® by the W. R. Grace & Co., Davison Chemical Division. Theprecipitated silica materials include those marketed by the J. M. HuberCorp. under the trade name Zeodent®, including the silica carrying thedesignation Zeodent 115 and 119. These silica abrasives are described inU.S. Pat. No. 4,340,583, to Wason, incorporated herein by reference.

In certain embodiments, abrasive materials useful in the practice of theoral care compositions in accordance with the invention include silicagels and precipitated amorphous silica having an oil absorption value ofabout less than 100 cc/100 g silica and in the range of about 45 cc/100g to about 70 cc/100 g silica. Oil absorption values are measured usingthe ASTA Rub-Out Method D281. In certain embodiments, the silicas arecolloidal particles having an average particle size of about 3 micronsto about 12 microns, and about 5 to about 10 microns.

In particular embodiments, the abrasive materials comprise very smallparticles, e.g. having a d50<5 microns. For example, small particlesilica (SPS) having a d50 of 3-4 microns, for example Sorbosil AC43®(Ineos). Such small particles are particularly useful in formulationstargeted at reducing hypersensitivity. The small particle component maybe present in combination with a second larger particle abrasive. Incertain embodiments, for example, the formulation comprises about 3-8%SPS and about 25-45% of a conventional abrasive.

Low oil absorption silica abrasives particularly useful in the practiceof the invention are marketed under the trade designation Sylodent XWA®by Davison Chemical Division of W.R. Grace & Co., Baltimore, Md. 21203.Sylodent 650 XWA® silica hydrogel composed of particles of colloidalsilica having a water content of 29% by weight averaging about 7 toabout 10 microns in diameter, and an oil absorption of less than about70 ccc/100 g of silica is an example of a low oil absorption silicaabrasive useful in the practice of the present invention. The abrasiveis present in the oral care composition of the present invention at aconcentration of about 10 to 60% by weight, in other embodiment about 20to 45% by weight, and in another embodiment about 30 to 50% by weight.

Agents to Increase the Amount of Foaming

The oral care compositions of the invention also may include an agent toincrease the amount of foam that is produced when the oral cavity isbrushed.

Illustrative examples of agents that increase the amount of foaminclude, but are not limited to polyoxyethylene and certain polymersincluding, but not limited to, alginate polymers.

The polyoxyethylene may increase the amount of foam and the thickness ofthe foam generated by the oral care carrier component of the presentinvention. Polyoxyethylene is also commonly known as polyethylene glycol(“PEG”) or polyethylene oxide. The polyoxyethylenes suitable for thisinvention will have a molecular weight of about 200,000 to about7,000,000. In one embodiment the molecular weight will be about 600,000to about 2,000,000 and in another embodiment about 800,000 to about1,000,000. Polyox® is the trade name for the high molecular weightpolyoxyethylene produced by Union Carbide.

The polyoxyethylene may be present in an amount of about 1% to about90%, in one embodiment about 5% to about 50% and in another embodimentabout 10% to about 20% by weight of the oral care carrier component ofthe oral care compositions of the present invention. The dosage offoaming agent in the oral care composition (i.e., a single dose) isabout 0.01 to about 0.9% by weight, about 0.05 to about 0.5% by weight,and in another embodiment about 0.1 to about 0.2% by weight.

Surfactants

Another agent optionally included in the oral care composition of theinvention is a surfactant or a mixture of compatible surfactants.Suitable surfactants are those which are reasonably stable throughout awide pH range, for example, anionic, cationic, nonionic or zwitterionicsurfactants.

Suitable surfactants are described more fully, for example, in U.S. Pat.No. 3,959,458, to Agricola et al.; U.S. Pat. No. 3,937,807, to Haefele;and U.S. Pat. No. 4,051,234, to Gieske et al., which are incorporatedherein by reference.

In certain embodiments, the anionic surfactants useful herein includethe water-soluble salts of alkyl sulfates having about 10 to about 18carbon atoms in the alkyl radical and the water-soluble salts ofsulfonated monoglycerides of fatty acids having about 10 to about 18carbon atoms. Sodium lauryl sulfate, sodium lauroyl sarcosinate andsodium coconut monoglyceride sulfonates are examples of anionicsurfactants of this type. Mixtures of anionic surfactants may also beutilized.

In another embodiment, cationic surfactants useful in the presentinvention can be broadly defined as derivatives of aliphatic quaternaryammonium compounds having one long alkyl chain containing about 8 toabout 18 carbon atoms such as lauryl trimethylammonium chloride, cetylpyridinium chloride, cetyl trimethylammonium bromide,di-isobutylphenoxyethyldimethylbenzylammonium chloride, coconutalkyltrimethylammonium nitrite, cetylpyridinium fluoride, and mixturesthereof.

Illustrative cationic surfactants are the quaternary ammonium fluoridesdescribed in U.S. Pat. No. 3,535,421, to Briner et al., hereinincorporated by reference. Certain cationic surfactants can also act asgermicides in the compositions.

Illustrative nonionic surfactants that can be used in the compositionsof the invention can be broadly defined as compounds produced by thecondensation of alkylene oxide groups (hydrophilic in nature) with anorganic hydrophobic compound which can be aliphatic or alkylaromatic innature. Examples of suitable nonionic surfactants include, but are notlimited to, the Pluronics, polyethylene oxide condensates of alkylphenols, products derived from the condensation of ethylene oxide withthe reaction product of propylene oxide and ethylene diamine, ethyleneoxide condensates of aliphatic alcohols, long chain tertiary amineoxides, long chain tertiary phosphine oxides, long chain dialkylsulfoxides and mixtures of such materials.

In certain embodiments, zwitterionic synthetic surfactants useful in thepresent invention can be broadly described as derivatives of aliphaticquaternary ammonium, phosphomium, and sulfonium compounds, in which thealiphatic radicals can be straight chain or branched, and wherein one ofthe aliphatic substituents contains about 8 to about 18 carbon atoms andone contains an anionic water-solubilizing group, e.g., carboxy,sulfonate, sulfate, phosphate or phosphonate. Illustrative examples ofthe surfactants suited for inclusion into the composition include, butare not limited to, sodium alkyl sulfate, sodium lauroyl sarcosinate,cocoamidopropyl betaine and polysorbate 20, and combinations thereof.

In a particular embodiment, the Composition of the Invention comprisesan anionic surfactant, e.g., sodium lauryl sulfate.

The surfactant or mixtures of compatible surfactants can be present inthe compositions of the present invention in about 0.1% to about 5.0%,in another embodiment about 0.3% to about 3.0% and in another embodimentabout 0.5% to about 2.0% by weight of the total composition.

Flavoring Agents

The oral care compositions of the invention may also include a flavoringagent. Flavoring agents which are used in the practice of the presentinvention include, but are not limited to, essential oils as well asvarious flavoring aldehydes, esters, alcohols, and similar materials.Examples of the essential oils include oils of spearmint, peppermint,wintergreen, sassafras, clove, sage, eucalyptus, marjoram, cinnamon,lemon, lime, grapefruit, and orange. Also useful are such chemicals asmenthol, carvone, and anethole. Certain embodiments employ the oils ofpeppermint and spearmint.

The flavoring agent is incorporated in the oral composition at aconcentration of about 0.1 to about 5% by weight and about 0.5 to about1.5% by weight. The dosage of flavoring agent in the individual oralcare composition dosage (i.e., a single dose) is about 0.001 to 0.05% byweight and in another embodiment about 0.005 to 0.015% by weight.

Chelating Agents

The oral care compositions of the invention also may optionally includeone or more chelating agents able to complex calcium found in the cellwalls of the bacteria. Binding of this calcium weakens the bacterialcell wall and augments bacterial lysis.

Another group of agents suitable for use as chelating agents in thepresent invention are the soluble pyrophosphates. The pyrophosphatesalts used in the present compositions can be any of the alkali metalpyrophosphate salts. In certain embodiments, salts include tetra alkalimetal pyrophosphate, dialkali metal diacid pyrophosphate, trialkalimetal monoacid pyrophosphate and mixtures thereof, wherein the alkalimetals are sodium or potassium. The salts are useful in both theirhydrated and unhydrated forms. An effective amount of pyrophosphate saltuseful in the present composition is generally enough to provide atleast 1.0 wt. % pyrophosphate ions, about 1.5 wt. % to about 6 wt. %,about 3.5 wt. % to about 6 wt. % of such ions.

Polymers

The oral care compositions of the invention also optionally include oneor more polymers, such as polyethylene glycols, polyvinylmethyl ethermaleic acid copolymers, polysaccharides (e.g. cellulose derivatives, forexample carboxymethyl cellulose, or polysaccharide gums, for examplexanthan gum or carrageenan gum). Acidic polymers, for examplepolyacrylate gels, may be provided in the form of their free acids orpartially or fully neutralized water soluble alkali metal (e.g.potassium and sodium) or ammonium salts. Certain embodiments include 1:4to 4:1 copolymers of maleic anhydride or acid with another polymerizableethylenically unsaturated monomer, for example, methyl vinyl ether(methoxyethylene) having a molecular weight (M.W.) of about 30,000 toabout 1,000,000. These copolymers are available for example as GantrezAN 139(M.W. 500,000), AN 119 (M.W. 250,000) and S-97 PharmaceuticalGrade (M.W. 70,000), of GAF Chemicals Corporation.

Other operative polymers include those such as the 1:1 copolymers ofmaleic anhydride with ethyl acrylate, hydroxyethyl methacrylate,N-vinyl-2-pyrollidone, or ethylene, the latter being available forexample as Monsanto EMA No. 1103, M.W. 10,000 and EMA Grade 61, and 1:1copolymers of acrylic acid with methyl or hydroxyethyl methacrylate,methyl or ethyl acrylate, isobutyl vinyl ether or N-vinyl-2-pyrrolidone.

Suitable generally, are polymerized olefinically or ethylenicallyunsaturated carboxylic acids containing an activated carbon-to-carbonolefinic double bond and at least one carboxyl group, that is, an acidcontaining an olefinic double bond which readily functions inpolymerization because of its presence in the monomer molecule either inthe alpha-beta position with respect to a carboxyl group or as part of aterminal methylene grouping. Illustrative of such acids are acrylic,methacrylic, ethacrylic, alpha-chloroacrylic, crotonic, beta-acryloxypropionic, sorbic, alpha-chlorsorbic, cinnamic, beta-styrylacrylic,muconic, itaconic, citraconic, mesaconic, glutaconic, aconitic,alpha-phenylacrylic, 2-benzyl acrylic, 2-cyclohexylacrylic, angelic,umbellic, fumaric, maleic acids and anhydrides. Other different olefinicmonomers copolymerizable with such carboxylic monomers includevinylacetate, vinyl chloride, dimethyl maleate and the like. Copolymerscontain sufficient carboxylic salt groups for water-solubility.

A further class of polymeric agents includes a composition containinghomopolymers of substituted acrylamides and/or homopolymers ofunsaturated sulfonic acids and salts thereof, in particular wherepolymers are based on unsaturated sulfonic acids selected fromacrylamidoalykane sulfonic acids such as 2-acrylamide 2 methylpropanesulfonic acid having a molecular weight of about 1,000 to about2,000,000, described in U.S. Pat. No. 4,842,847, Jun. 27, 1989 to Zahid,incorporated herein by reference.

Another useful class of polymeric agents includes polyamino acids,particularly those containing proportions of anionic surface-activeamino acids such as aspartic acid, glutamic acid and phosphoserine, asdisclosed in U.S. Pat. No. 4,866,161 Sikes et al., incorporated hereinby reference.

In preparing oral care compositions, it is sometimes necessary to addsome thickening material to provide a desirable consistency or tostabilize or enhance the performance of the formulation. In certainembodiments, the thickening agents are carboxyvinyl polymers,carrageenan, hydroxyethyl cellulose and water soluble salts of celluloseethers such as sodium carboxymethyl cellulose and sodium carboxymethylhydroxyethyl cellulose. Natural gums such as karaya, gum arabic, and gumtragacanth can also be incorporated. Colloidal magnesium aluminumsilicate or finely divided silica can be used as component of thethickening composition to further improve the composition's texture. Incertain embodiments, thickening agents in an amount of about 0.5% toabout 5.0% by weight of the total composition are used.

Enzymes

The oral care compositions of the invention may also optionally includeone or more enzymes. Useful enzymes include any of the availableproteases, glucanohydrolases, endoglycosidases, amylases, mutanases,lipases and mucinases or compatible mixtures thereof. In certainembodiments, the enzyme is a protease, dextranase, endoglycosidase andmutanase. In another embodiment, the enzyme is papain, endoglycosidaseor a mixture of dextranase and mutanase. Additional enzymes suitable foruse in the present invention are disclosed in U.S. Pat. No. 5,000,939 toDring et al., U.S. Pat. No. 4,992,420; U.S. Pat. No. 4,355,022; U.S.Pat. No. 4,154,815; U.S. Pat. No. 4,058,595; U.S. Pat. No. 3,991,177;and U.S. Pat. No. 3,696,191 all incorporated herein by reference. Anenzyme of a mixture of several compatible enzymes in the currentinvention constitutes about 0.002% to about 2.0% in one embodiment orabout 0.05% to about 1.5% in another embodiment or in yet anotherembodiment about 0.1% to about 0.5%.

Water

Water may also be present in the oral compositions of the invention.Water, employed in the preparation of commercial oral compositionsshould be deionized and free of organic impurities. Water commonly makesup the balance of the compositions and includes about 10% to about 90%,about 20% to about 60% or about 10% to about 30% by weight of the oralcompositions. This amount of water includes the free water which isadded plus that amount which is introduced with other materials such aswith sorbitol or any components of the invention.

Humectants

Within certain embodiments of the oral compositions, it is alsodesirable to incorporate a humectant to prevent the composition fromhardening upon exposure to air. Certain humectants can also impartdesirable sweetness or flavor to dentifrice compositions. The humectant,on a pure humectant basis, generally includes about 15% to about 70% inone embodiment or about 30% to about 65% in another embodiment by weightof the dentifrice composition.

Suitable humectants include edible polyhydric alcohols such asglycerine, sorbitol, xylitol, propylene glycol as well as other polyolsand mixtures of these humectants. Mixtures of glycerine and sorbitol maybe used in certain embodiments as the humectant component of thetoothpaste compositions herein.

In addition to the above described components, the embodiments of thisinvention can contain a variety of optional dentifrice ingredients someof which are described below. Optional ingredients include, for example,but are not limited to, adhesives, sudsing agents, flavoring agents,sweetening agents, additional antiplaque agents, abrasives, and coloringagents. These and other optional components are further described inU.S. Pat. No. 5,004,597, to Majeti; U.S. Pat. No. 3,959,458 to Agricolaet al. and U.S. Pat. No. 3,937,807, to Haefele, all being incorporatedherein by reference.

Stimulation of Antimicrobial Peptides:

Human CAP-18, a human cathelicidin, was first identified in neutrophilsand later shown to be expressed in various squamous epithelia, surfaceepithelial cells of the conducting airways, and serous and mucous cellsof the submucosal glands, by keratinocytes in inflamed skin and byspecific lymphocyte and monocyte populations. Human CAP-18 is the onlyhuman cathelicidin identified to date. It belongs to the cathelicidinfamily of antimicrobial peptides that are characterized by a conservedN-terminal cathelin domain and a variable C-terminal antimicrobialdomain. This C-terminal domain can be cleaved off from the precursor byproteinases, releasing the active peptide. Exocytosed material fromneutrophils contains hCAP-18 that has been proteolytically cleaved byproteinase-3 yielding the 4.5 kD active alpha helical peptide LL-37.LL-37 displays antimicrobial activity against a broad spectrum ofmicroorganisms and possesses synergistic antibacterial effects withother antimicrobial peptides, such as defensins. It is believed thatcathelicidins play a role in effective host defense against infection. Astudy has that mice deficient in the murine cathelicidin-relatedantimicrobial peptide suffer from more severe bacterial skin infections.Tjabring a, Sandra, et al., J. Immunol, 171:6690-6696 (2003). Foranother instance, deficiency in saliva LL-37 accords with occurrence ofperiodontal disease in patients with morbus Kostmann. Pütsep, Katrin, etal, Lancet, 360:1144-1149 (2002).

As used herein hCAP-18 is considered to be the pro-form of LL-37. Aftercleavage by proteinases, a 4 kD polypeptide results as the active formof LL-37. As used herein, both forms are referred to as LL-37. Theantibodies that bind to both forms of the LL-37, the pro-form and theactive form are described in corresponding patent application Ser. No.(Attorney docket no: 0024.0012-001) filed on even date herewith,entitled “New Methods of Making An Antibody and Compositions Thereof” byToshihisa Kawai, et al., the entire teachings of which are incorporatedherein by reference. LL-37 is one of the antimicrobial peptides used forthe present invention. Examples of anti-microbial peptides includeLL-37, hBD2, hBD3, Ghrelin, Lysozyme or a combination thereof.

It has been determined that LL-37, hBD2, hBD3, Ghrelin, Lysozyme hasantimicrobial properties. For example, LL-37 is stimulated by certaincompounds such as those having trihydroxybenzoate moieties. Inparticular, trihydroxybenzoate derivatives including3,4,5-trihydroxybenzoic acid (i.e., gallic acid), its isoform,2,3,4-trihydroxybenzoic acid, C₁₋₄alkyl 3,4,5-trihydroxybenzoate (e.g.,ethyl 3,4,5-trihydroxybenzoate), C₁₋₄alkyl 2,3,4-trihydroxybenzoate(e.g., ethyl 2,3,4-trihydroxybenzoate), EGCG and mixtures thereof. Inparticular, “THBC” refers to chemical structures such astrihydroxybenzoates that comprise tri-hydroxy benzoic acid and itsisoforms, which are found in the polyphenol extracts of natural plantproducts or in antioxidant food preservatives. As suchtrihydroxybenzoate moieties can be used as a positive control the LL-37,hBD2, hBD3, Ghrelin, Lysozyme assay described herein.

Compounds for stimulating production of anti-microbial peptides can behelpful in treating bacterial infection. Particularly, such compositionsare particularly useful for treating a bacterial infection wherein (a)the bacteria is antibiotic resistant, (b) the patient is allergic toantibiotics; (c) the patient is immuno-compromised (e.g., HIV/AIDSpatients); or (d) the infection is of an oral cavity. Additionally,LL-37, hBD2, hBD3, Ghrelin, Lysozyme are a potent antimicrobial againstvarious staphylococcal species and particularly virulentantibiotic-resistant strains of pathogenic bacteria, such as those foundin Methicilin-resistant Staphylococcus aureus (MRSA). Such compounds canalso be useful for 1) oral health care products, 2) skin and mucosalhealth care products, 3) antimicrobial drugs, and 4) neutraceuticalproducts.

Antibodies for Assessing Anti-Microbial Peptides:

Antibodies that are specific to LL-37, hBD2, hBD3, Ghrelin, Lysozyme ora combination thereof are helpful in determining compounds thatstimulate antimicrobial peptide production, and would be helpful intreating the antibacterial disease or conditions. Compounds thatstimulate antimicrobial peptide production can be included incompositions in which anti-microbial properties are needed. Since it isalso reported that LL-37 can down regulate the production ofpro-inflammatory cytokines, the increased production of LL-37 alsoresults in the suppression of inflammatory responses. Scott M G, J.Immunol. 2002; 169:3883-389 (2002). Compounds that stimulate LL-37 hBD2,hBD3, Ghrelin, and/or Lysozyme can be included in health compositions(e.g., toothpaste, mouth wash, floss) and medications to treat variousdisease, including those related to the gut (e.g., for Crohn's diseaseor Colitis), and the like.

Antibodies can be used to assess the stimulation of antimicrobialpeptide production. The term, “antibody,” encompasses polyclonalantibodies, monoclonal antibodies, single chain antibodies, VHHantibodies, chimeric, humanized, primatized, CDR-grafted, and veneeredantibodies. This term further includes portions derived from differentspecies, human antibodies which are native or derived from combinatoriallibraries, and the like. Conventional techniques can chemically jointogether the various portions of these antibodies. Genetic engineeringtechniques can also prepare the antibody as a contiguous protein. Forexample, nucleic acids encoding a chimeric or humanized chain can beexpressed to produce a contiguous protein.

Specifically, the term “antibody fragment” refers to portion of animmunoglobulin having specificity to the LL-37 hBD2, hBD3, Ghrelin,Lysozyme or a portion thereof. The term, “antibody fragment”, isintended to encompass fragments from both polyclonal and monoclonalantibodies including transgenically produced antibodies, single-chainantibodies (scFvs), recombinant Fabs, and recombinant camelidheavy-chain-only antibodies (VHHs). VHHs are also referred to asnanobodies.

Functional fragments of antibodies, including fragments of chimeric,humanized, primatized, veneered or single chain antibodies, can also beproduced. Functional fragments or portions of the foregoing antibodiesinclude those which are reactive with the LL-37, hBD2, hBD3, Ghrelin,Lysozyme. For example, antibody fragments capable of binding to LL-37,hBD2, hBD3, Ghrelin, Lysozyme or portion thereof, including, but notlimited to scFvs, Fabs, VHHs, Fv, Fab, Fab′ and F(ab′)2 are encompassedby the invention. Such fragments can be produced by enzymatic cleavageor by recombinant techniques. For instance, papain or pepsin cleavagecan generate Fab or F(ab′)2 fragments, respectively. Antibodies can alsobe produced in a variety of truncated forms using antibody genes inwhich one or more stop codons has been introduced upstream of thenatural stop site. For example, a chimeric gene encoding a F(ab′)2 heavychain portion can be designed to include DNA sequences encoding the CHIdomain and hinge region of the heavy chain.

The term “antibody” also includes various forms of modified antibodies.For example, modification may occur by directly or indirectly attachinga detectable label. The detectable labels may include a radioisotope,spin label, antigen label such as a FLAG tag, enzyme label, fluorescentor chemiluminescent group and the like.

The term “sample” includes fluid and/or cells from an oral swab (e.g.,containing saliva and/or epithelial cells from the oral cavity), tissue,fluid, whole blood, plasma, serum and aqueous blood components from apatient.

Methods of Assessment of Anti-Microbial Peptide Production and Methodsof Making an Anti-Microbial Peptide Antibody:

Method for assessing the presence, absence or level of LL-37, hBD2,hBD3, Ghrelin, Lysozyme, in a sample, is encompassed by the presentinvention. Suitable assays include immunological methods, such as highthroughput assays, radioimmunoassay, enzyme-linked immunosorbent assays(ELISA), chemiluminescence assays, and rapid immunochromatographicassays. A high throughput assays is a preferred embodiment of thepresent invention. Any method known now or developed later can be usedfor measuring LL-37, hBD2, hBD3, Ghrelin, or Lysozyme using theantibodies specific to these peptides.

In several of the preferred embodiments, immunological techniques detectthe presence, absence, or levels of LL-37, hBD2, hBD3, Ghrelin, orLysozyme described herein by means of an anti-LL-37, anti-hBD2,anti-hBD3, anti-Ghrelin, or anti-Lysozyme antibody (i.e., one or moreantibodies). The term antibody includes one or more monoclonalantibodies or fragments thereof, and mixtures or cocktails thereof.

Anti-LL-37, anti-hBD2, anti-hBD3, anti-Ghrelin, or anti-Lysozymeantibody antibodies can be raised against appropriate immunogens, suchas isolated and/or recombinant anti-microbial polypeptides describedherein, analogs or portion thereof (including synthetic molecules, suchas synthetic peptides).

To increase immunogenicity, the LL-37 protein underwent a chemicalmodification prior to being injected into an animal (e.g., mouse,rodent, rabbit, goat, monkey, camel, and the like) during the monoclonalantibody procedures. The chemical modification allows for a reactionwith certain amino acids present in the protein. In this case,methylglyoxal was used to react with Arginine (R) and Lysine (K) presentin LL-37. When methylglyoxal was combined with LL-37, the absorption byantigen presenting cells increased to thereby increase immunogenicityand develop a LL-37 specific antibody having a high affinity. Antigenpresenting cells express the specific receptor (RAGE; the receptor foradvanced glycation end products) for methylglyoxal-modified peptide andthereby allow for better absorption. Methylglyoxal is contacted with theantigen to be modified in an amount between about 1 nM and about 1 mM.This procedures can be applied to any of the anti-microbial peptideshaving between 1 and 30% total content of arginine, Lysine, or cysteine(C).

In one embodiment, antibodies are raised against an isolated,recombinant, active, chemically modified of LL-37, hBD2, hBD3, Ghrelin,or Lysozyme polypeptide described herein or portion thereof (e.g., apeptide) or against a host cell which expresses recombinant orchemically modified of LL-37, hBD2, hBD3, Ghrelin, or Lysozyme. Inaddition, cells expressing recombinant or modified antigenic of LL-37,hBD2, hBD3, Ghrelin, or Lysozyme polypeptides described herein, such astransfected cells, can be used as immunogens or in a screen for antibodywhich binds receptor.

Preparing the immunizing antigen can be done, as described above and anysuitable technique, now known or later developed, can be used to producepolyclonal or monoclonal antibodies. The art contains a variety of thesemethods (see e.g., Kohler et al., Nature, 256: 495-497 (1975) and Eur.J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266: 550-552(1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D.Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y.); Current Protocols In MolecularBiology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al.,Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)).Generally, fusing a suitable immortal or myeloma cell line, such asSP2/0, with antibody producing cells can produce a hybridoma. Animalsimmunized with the antigen of interest, after chemical modification,provide a cell that produces an antibody. Surprisingly, one or moreantibodies specific for the native antigen are made and have a highbinding affinity to the native antigen, even though the immunizingantigen was chemically modified. The immunized animal generally producescells that make antibody that is specific to the antigen and these cellsare typically cells from the spleen or lymph nodes. Cells from thespleen or lymph node are teased or separated from one another andcultured. Polyethylene glycol (PEG) or similar compound is used to fusethe myeloma cell line with the spleen or lymph node cells and aselective medium is used in which only fused cells can grow. Selectiveculture conditions isolate antibody producing hybridoma cells whilelimiting dilution techniques produce them. This mixture of cells isdiluted and clones are grown from single parent cells generally in wellsof a plate. The antibodies secreted by the different clones are thenassayed for their ability to bind to the antigen. Researchers can usesuitable assays such as ELISA to select antibody producing cells withthe desired specificity. The hybridomas can be grown indefinitely in asuitable cell culture media, or they can be injected in mice into theperitoneal cavity, wherein they produce tumors containing anantibody-rich fluid called ascites fluid. The ascites that contains theantibody can be drained, bled or otherwise withdrawn from the animal.The antibody can be purified using standard purification techniques(e.g., ultra-filtration, dialysis, and chromatography).

Other suitable methods can produce or isolate antibodies of therequisite specificity. Examples of other methods include selectingrecombinant antibody from a library or relying upon immunization oftransgenic animals such as mice to make human monoclonal antibodies.

According to the method, an assay can determine the presence, absence orlevel of LL-37, hBD2, hBD3, Ghrelin, or Lysozyme peptides in abiological sample. Such an assay includes combining the sample to betested with an antibody having specificity for LL-37, hBD2, hBD3,Ghrelin, or Lysozyme described herein, under conditions suitable forformation of a complex between antibody and LL-37, hBD2, hBD3, Ghrelin,or Lysozyme, and detecting or measuring (directly or indirectly) theformation of a complex. In the event that a compound is being assessed,the sample can be combined with the compound to be tested and incubatedunder conditions to allow for expression of LL-37, hBD2, hBD3, Ghrelin,or Lysozyme. The sample can be obtained directly or indirectly (e.g.,provided by a healthcare provider), and can be prepared by a methodsuitable for the particular sample (e.g., saliva, epithelial cells fromthe oral cavity, urine, sputum, fecal matter, cerebral spinal fluid,whole blood, platelet rich plasma, platelet poor plasma, serum) andselect an assay format. Methods of combining sample and antibody, andmethods of detecting complex formation are also selected to becompatible with the assay format.

The assays described herein can be modified to assess the effect of acompound on LL-37, hBD2, hBD3, Ghrelin, or Lysozyme expression. In sucha case, the compound can be introduced to the assay by contacting thecompound with cells or samples that can express LL-37, hBD2, hBD3,Ghrelin, or Lysozyme. The cells or sample can be subjected to compoundto be tested to determine if the compound has an effect on LL-37, hBD2,hBD3, Ghrelin, or Lysozyme expression. Alternatively, the subject, fromwhom the sample is taken, can be subjected to the compound and then asample is taken. The sample can then be assessed for LL-37, hBD2, hBD3,Ghrelin, or Lysozyme expression, as described herein. Since it has beendetermined that trihydroxybenzoate stimulates LL-37, hBD2, hBD3,Ghrelin, or Lysozyme expression, a trihydroxybenzoate moiety can be usedas a positive control in the assay.

Suitable labels can be detected directly, such as radioactive,fluorescent or chemiluminescent labels. They can also be indirectlydetected using labels such as enzyme labels and other antigenic orspecific binding partners like biotin. Examples of such labels includefluorescent labels such as fluorescein, rhodamine, chemiluminescentlabels such as luciferase, radioisotope labels such as 32P, 125I, 131I,enzyme labels such as horseradish peroxidase, and alkaline phosphatase,galactosidase, biotin, avidin, spin labels and the like. The detectionof antibodies in a complex can also be done immunologically with asecond antibody, which is then detected (e.g., by means of a label).Conventional methods or other suitable methods can directly orindirectly label an antibody. Labeled primary and secondary antibodiescan be obtained commercially or prepared using methods know to one ofskill in the art (see Harlow, E. and D. Lane, 1988, Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor,N.Y.).

In a preferred embodiment, the presence, absence, or level of LL-37 in asample is determined using a high throughput assay, an ELISA assay, asandwich ELISA assay, or immunochromatographic assay.

For detection of LL-37, hBD2, hBD3, Ghrelin, or Lysozyme in a suitablesample, a sample (e.g., saliva) is collected. Samples can be processedas known in the art. The compound to be assessed can be contacted withthe sample, or in contact with the subject prior to taking the sample.For example, the compound can be part of an oral composition that thesubject uses.

In an embodiment, LL-37, hBD2, hBD3, Ghrelin, or Lysozyme is determinedusing an ELISA assay or a sandwich ELISA assay. In one embodiment,murine L2 is used as capture antibody and murine L7 is used as detectorantibody.

In an embodiment, the assay further includes combining a suitablesample, and optionally the compound to be tested, with a compositionhaving an anti-LL-37, anti-hBD2, anti-hBD3, anti-Ghrelin, oranti-Lysozyme polypeptide antibody as detector (e.g., biotinylatedanti-microbial polypeptides MAb and HRP-streptavidin, or HRP-conjugatedanti-microbial polypeptides Mab), and a solid support, such as amicrotiter plate, having an anti-microbial polypeptide capture antibodybound (directly or indirectly) thereto. The detector antibody binds to adifferent anti-microbial polypeptide epitope from that recognized by thecapture antibody, under conditions suitable for the formation of thecomplex. The assay then involves determining the formation of complex inthe samples. The presence of or increase in LL-37, hBD2, hBD3, Ghrelin,or Lysozyme in a sample of an individual indicates the presence of acompound that increased expression or production of LL-37, hBD2, hBD3,Ghrelin, or Lysozyme, whereas the absence of or decrease in a LL-37,hBD2, hBD3, Ghrelin, or Lysozyme polypeptide indicates the compound tobe tested that does not increase expression or production.

The solid support, such as a microtiter plate, dipstick, bead, pad,strip, or other suitable support, can be coated directly or indirectlywith an anti-LL-37, anti-hBD2, anti-hBD3, anti-Ghrelin, or anti-Lysozymepolypeptide antibody or antimicrobial specific antigen. For example, ananti-LL-37, anti-hBD2, anti-hBD3, anti-Ghrelin, or anti-Lysozymepolypeptide antibody can coat a microtiter well, or a biotinylatedanti-LL-37, anti-hBD2, anti-hBD3, anti-Ghrelin, or anti-Lysozymepolypeptide Mab can be added to a streptavidin coated support. Withrespect to a immunochromatographic assay, a pad or strip can be coatedwith an antibody specific for the antigen, and when a sample having theone or more of antigens described herein comes into contact with theantibody, the complex can turn a color with aid of a detector, asfurther described herein. A variety of immobilizing or coating methodsas well as a number of solid supports can be used, and can be selectedaccording to the desired format.

In another embodiment, the sample (or an LL-37, hBD2, hBD3, Ghrelin, orLysozyme polypeptide standard) is combined with the solid supportsimultaneously with the detector antibody, and optionally with a one ormore reagents by which detection is monitored. For example, the samplecan be combined with the solid support simultaneously with (a)HRP-conjugated anti-LL-37 polypeptide Mab, or (b) a biotinylatedanti-LL-37, hBD2, hBD3, Ghrelin, or Lysozyme polypeptide Mab andHRP-streptavidin.

A known amount of an LL-37, hBD2, hBD3, Ghrelin, or Lysozyme polypeptidestandard can be prepared and processed as described above for a suitablesample. This LL-37, hBD2, hBD3, Ghrelin, or Lysozyme polypeptidestandard assists in quantifying the amount of LL-37 detected bycomparing the level of LL-37, hBD2, hBD3, Ghrelin, or Lysozyme in thesample relative to that in the standard. In one embodiment, activeLL-37, hBD2, hBD3, Ghrelin, or Lysozyme is used as a standard.

A physician, technician, apparatus or a qualified person can compare theamount of detected complex with a suitable control to determine if theLL-37, hBD2, hBD3, Ghrelin, or Lysozyme levels are elevated or not. Acontrol can be the level of LL-37, hBD2, hBD3, Ghrelin, or Lysozyme in asample take from the subject, but not subjected to the compound to betested. A positive control can be the level of LL-37, hBD2, hBD3,Ghrelin, or Lysozyme subjected to a known compound that stimulatesLL-37, hBD2, hBD3, Ghrelin, or Lysozyme expression (e.g.,trihydroxybenzoate moiety, or EGCG). A control can also be the averagelevel of LL-37, hBD2, hBD3, Ghrelin, or Lysozyme for the particularsample, in a healthy population.

Typical assays for LL-37, hBD2, hBD3, Ghrelin, or Lysozyme aresequential assays in which a plate is coated with first antibody, sampleis added, the plate is washed, second tagged antibody is added, and theplate is washed and bound second antibody is quantified. In anotherembodiment, a format in which antibodies and the sample are addedsimultaneously, in a competitive ELISA format, can achieve greatersensitivity.

A variety of methods can determine the amount of LL-37, hBD2, hBD3,Ghrelin, or Lysozyme in complexes. For example, when HRP is used as alabel, a suitable substrate such as OPD can be added to produce colorintensity directly proportional to the bound anti-LL-37, anti-hBD2,anti-hBD3, anti-Ghrelin, or anti-Lysozyme polypeptides mAb (assessede.g., by optical density), and therefore to the LL-37, hBD2, hBD3,Ghrelin, or Lysozyme in the sample.

A technician, physician, qualified person or apparatus can compare theresults to a suitable control such as a standard, or baseline levels ofLL-37, hBD2, hBD3, Ghrelin, or Lysozyme in a sample from the same donor.For example, the assay can be performed using a known amount of LL-37,hBD2, hBD3, Ghrelin, or Lysozyme standard in lieu of a sample, and astandard curved established. One can relatively compare known amounts ofthe LL-37, hBD2, hBD3, Ghrelin, or Lysozyme standard to the amount ofcomplex formed or detected.

The methods described herein for assaying compounds to determineantibacterial (e.g., antimicrobial) peptide stimulation, can also beused for assessing compounds for their ability to bind DDX5. SeeExemplification. An antibody specific to DDX5 can be made and used inassays described herein. Compounds that stimulate the anti-microbialpeptides bind to or have affinity to DDX5.

In an embodiment, methods for stimulating the production ofantimicrobial peptides include contacting a mammalian cell with atrihydroxybenzoic acid derivative, and assessing the level ofstimulation of the production of anti-microbial peptides. Samples ofcells can be taken, as described herein, and contacted with atrihydroxybenzoate derivative in amounts described herein. Theassessment of the level of antimicrobial peptides can be done using theassays and antibodies described herein.

Another embodiment of the invention includes stimulating the productionof antimicrobial peptide in an individual. The method involves selectingan individual having a decreased level of antimicrobial peptidestimulation, as compared to a control. Samples from the individual canbe taken, as described herein, and assayed to determine the level ofantimicrobial peptide stimulation. The level can be compared to a priorlevel from the individual, or to a level from a healthy individual. Inthe event that the level is decreased, as compared to the control, asdefined herein, then an amount of a compound that stimulates productionof anti-microbial peptides can be administered. Various amounts andcompositions are described here. The level can be re-assessed todetermine if the level increase after administration.

Anti-LL-37, anti-hBD2, anti-hBD3, anti-Ghrelin, or anti-Lysozymepolypeptides mAb antibody can be produced recombinantly using a DNAsequence that encodes the antigen, which has been inserted into anexpression vector and expressed in an appropriate host cell. DNAsequences encoding an anti-LL-37, anti-hBD2, anti-hBD3, anti-Ghrelin, oranti-Lysozyme antibody can, for example, be identified by assaying theantibody or fragment thereof against LL-37, hBD2, hBD3, Ghrelin, orLysozyme and assessing binding and/or affinity characteristics.

The assays of the present invention include nucleic acid molecules(e.g., probes or primers) that hybridize to the anti-LL-37, anti-hBD2,anti-hBD3, anti-Ghrelin, or anti-Lysozyme antibody sequences, under highor moderate stringency conditions. In one aspect, the present inventionincludes molecules that are or hybridize to at least about 20 contiguousnucleotides or longer in length (e.g., 50, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000,3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000). Suchmolecules hybridize to one of anti-microbial peptides antibody nucleicacid sequences under high stringency conditions. The present inventionincludes such molecules and those that encode a polypeptide that has thefunctions or biological activity described herein.

Typically the nucleic acid probe comprises a nucleic acid sequence andis of sufficient length and complementarity to specifically hybridize toa nucleic acid sequence that encodes anti-LL-37 anti-hBD2, anti-hBD3,anti-Ghrelin, or anti-Lysozyme polypeptide. For example, a nucleic acidprobe can be at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%or 90% the length of the anti-LL-37, anti-hBD2, anti-hBD3, anti-Ghrelin,or anti-Lysozyme antibody nucleic acid sequence. The requirements ofsufficient length and complementarity can be easily determined by one ofskill in the art. Suitable hybridization conditions (e.g., highstringency conditions) are also described herein. Additionally, thepresent invention encompasses fragments of the polypeptides of thepresent invention or nucleic acid sequences that encodes a polypeptidewherein the polypeptide has the biologically activity of the anti-LL-37polypeptides recited herein.

Stringency conditions for hybridization refers to conditions oftemperature and buffer composition which permit hybridization of a firstnucleic acid sequence to a second nucleic acid sequence, wherein theconditions determine the degree of identity between those sequenceswhich hybridize to each other. Therefore, “high stringency conditions”are those conditions wherein only nucleic acid sequences which are verysimilar to each other will hybridize. The sequences can be less similarto each other if they hybridize under moderate stringency conditions.Still less similarity is needed for two sequences to hybridize under lowstringency conditions. By varying the hybridization conditions from astringency level at which no hybridization occurs, to a level at whichhybridization is first observed, conditions can be determined at which agiven sequence will hybridize to those sequences that are most similarto it. The precise conditions determining the stringency of a particularhybridization include not only the ionic strength, temperature, and theconcentration of destabilizing agents such as formamide, but alsofactors such as the length of the nucleic acid sequences, their basecomposition, the percent of mismatched base pairs between the twosequences, and the frequency of occurrence of subsets of the sequences(e.g., small stretches of repeats) within other non-identical sequences.Washing is the step in which conditions are set so as to determine aminimum level of similarity between the sequences hybridizing with eachother. Generally, from the lowest temperature at which only homologoushybridization occurs, a 1% mismatch between two sequences results in a1° C. decrease in the melting temperature (Tm) for any chosen SSCconcentration. Generally, a doubling of the concentration of SSC resultsin an increase in the Tm of about 17° C. Using these guidelines, thewashing temperature can be determined empirically, depending on thelevel of mismatch sought. Hybridization and wash conditions areexplained in Current Protocols in Molecular Biology (Ausubel, F. M. etal., eds., John Wiley & Sons, Inc., 1995, with supplemental updates) onpages 2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.

High stringency conditions can employ hybridization at either (1) 1×SSC(10×SSC=3 M NaCl, 0.3 M Na₃-citrate.2H20 (88 g/liter), pH to 7.0 with 1M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calfthymus DNA at 65° C., (2) 1×SSC, 50% formamide, 1% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 42° C., (3) 1% bovine serum albumin(fraction V), 1 mM Na₂ . . . EDTA, 0.5 M NaHP04 (pH 7.2) (1M NaHP04=134g Na2HP04 . . . 7H20 , 4 ml 85% H3P04 per liter), 7% SDS, 0.1-2 mg/mldenatured calf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 MTris-HCl (pH 7.6), IX Denhardt's solution (100X=10 g Ficoll 400, 10 gpolyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymusDNA at 42° C., (5) 5×SSC, 5×Denhardt's solution, 1% SDS, 100 μg/mldenatured calf thymus DNA at 65° C., or (6) 5×SSC, 5×Denhardt'ssolution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at42° C., with high stringency washes of either (1) 0.3-0.1×SSC, 0.1% SDSat 65° C., or (2) 1 mM Na2EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS at 65° C.The above conditions are intended to be used for DNA-DNA hybrids of 50base pairs or longer. Where the hybrid is believed to be less than 18base pairs in length, the hybridization and wash temperatures should be5-10° C. below that of the calculated Tm of the hybrid, where Tm in °C.=(2× the number of A and T bases)+(4× the number of G and C bases).For hybrids believed to be about 18 to about 49 base pairs in length,the Tm in o C.=(81.5° C.+16.6(log 10M)+0.41(% G+C)−0.61 (%formamide)−500/L), where “M” is the molarity of monovalent cations(e.g., Na+), and “L” is the length of the hybrid in base pairs.

Methods of Treatment:

The present invention relates to methods for treating oral cavitydiseases or conditions in individuals by administering an amount of ananti-microbial peptide stimulation compound. The present invention alsorelates to methods for treating bacterial infections in an individual.In particular, the present invention relates to treating and/orpreventing diseases in an individual by anti-microbial peptidestimulation compound administration. To treat an individual with anoral/bacterial disease or condition means to alleviate, ameliorate orreduce the severity of one or more of symptoms associated with theoral/bacterial disease or condition. Prevention of a disease orcondition refers to delaying or suppressing the onset of the one or moresymptoms of the disease or condition. Additionally, the presentinvention encompasses reducing the severity of one or more symptomsassociated with the oral/bacterial disease or condition, which refers tominimizing the extent of one or more such symptoms that are experiencedby the individual.

Diseases or conditions, including oral disease or conditions, includedin the methods of the present invention are those that are caused bybacterial infection. Such diseases are known in the art and includee.g., thrush, gingivitis, periodontitis, oral candidiasis, salivarydiseases associated with bacterial, viral, or fungal infections; Crohn'sdisease, irritable bowel syndrome, ulcerative colitis, Helicobacterpylori caused stomach ulcer, and gastrointestinal diseases associatedwith mucosal bacteria. Additional examples of such infections includeMethicillin-resistant staphylococcus aureus (MRSA), pneumonia,meningitis, urinary tract infections, sinusitis, gastritis and the like.

Methods for determining if an individual has such a disease includediagnostic tests and evaluations that are known in the art. Bacterialdiseases or infections can be determined by taking a sample from theindividual and culturing the sample to determine if the bacterial grows.

Modes and Manner of Administration, Dosages

Compounds that stimulate anti-microbial peptide production areadministered for use with the methods described herein. Examples of suchcompounds include trihydroxybenzoate derivatives, as described in detailherein.

Such compounds used in the present invention can be administered with orwithout a carrier. The terms “pharmaceutically acceptable carrier” or a“carrier” refer to any generally acceptable excipient or drug deliverycomposition that is relatively inert and non-toxic. Exemplary carriersinclude sterile water, salt solutions (such as Ringer's solution),alcohols, gelatin, talc, viscous paraffin, fatty acid esters,hydroxymethylcellulose, polyvinyl pyrolidone, calcium carbonate,carbohydrates (such as lactose, sucrose, dextrose, mannose, albumin,starch, cellulose, silica gel, polyethylene glycol (PEG), dried skimmilk, rice flour, magnesium stearate, and the like. Suitableformulations and additional carriers are described in Remington'sPharmaceutical Sciences, (17th Ed., Mack Pub. Co., Easton, Pa.). Suchpreparations can be sterilized and, if desired, mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,preservatives and/or aromatic substances and the like which do notdeleteriously react with the active compounds. Typical preservatives caninclude; potassium sorbate, sodium metabisulfite, methylparaben, propylparaben, thimerosal, chloral derivative, etc. A carrier (e.g., apharmaceutically acceptable carrier) is preferred, but not necessary toadminister the compound.

The anti-microbial peptide stimulation compound can be a liquidsolution, suspension, emulsion, tablet, pill, capsule, sustained releaseformulation, or powder. The method of administration can dictate how thecomposition will be formulated. For example, the composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc.

The anti-microbial peptide stimulation compound used in the inventioncan be administered intravenously, parenterally, intramuscular,subcutaneously, orally (e.g., Cenestin, Estinyl, Estrace, Menest, Ogen,Premarin), nasally, topically (e.g., Estrace, Ogen, Ortho Dienestrol,Premarin), by inhalation, by implant (temporarily, e.g., Estring,Femring), by injection, by suppository (Vagifem) or transdermally (e.g.,Alora, Climara, Esclim, Estraderm, Vivelle, Vivelle-Dot). Thecomposition can be administered in a single dose or in more than onedose over a period of time to confer the desired effect (e.g.,periodically, daily, weekly, monthly, yearly, etc.). In one embodiment,anti-microbial peptide stimulation compound, in particular to beadministered has about 0.0001% to about 2% by weight of a Camelliaextract, and the extract includes trihydroxybenzoate derivatives in aconcentration from about 0.1 ug/ml to about 100 ug/ml.

The actual effective amounts of compound or drug can vary according tothe specific composition being utilized, the mode of administration andthe age, weight and condition of the patient. For example, as usedherein, an effective amount of the drug is an amount which stimulatesanti-microbial peptide production. Dosages for a particular individualpatient can be determined by one of ordinary skill in the art usingconventional considerations, (e.g. by means of an appropriate,conventional pharmacological protocol).

For enteral or mucosal application (including via oral and nasalmucosa), particularly suitable are tablets, liquids, drops,suppositories or capsules. A syrup, elixir or the like can be usedwherein a sweetened vehicle is employed. Liposomes, microspheres, andmicrocapsules are available and can be used.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Inparticular, carriers for parenteral administration include aqueoussolutions of dextrose, saline, pure water, ethanol, glycerol, propyleneglycol, peanut oil, sesame oil, polyoxyethylene-polyoxypropylene blockpolymers, and the like. Ampules are convenient unit dosages.

EXAMPLES

The following examples illustrate an oral composition of the describedinvention as compared to other formulations of similar compositions.Unless otherwise specified, all percentages are by weight.

Example 1 Induction of LL-37 Peptides

Induction of LL-37 and hBD2 mRNA Expression in Human Gingival EpithelialCells.

Both LL-37 and hBD2 mRNA expression in OBA9 cells are induced after a4-hour exposure to green tea polyphenol extract, Teaflan 90S (EGCG 50%,ECG 13%), and purified EGCG (95% pure, SIGMA) at concentrations of 0.1,1.0 and 10 ug/ml. EGCG increases the production of LL-37 and hBD2 at themRNA level in the human gingival epithelial cell line OBA9 (FIG. 1) aswell as the human lung carcinoma cell line A549.

In Vitro Induction of LL-37 Protein Expression in Human GingivalEpithelial Cells.

Using ELISA systems to detect LL-37 (Forsyth in-house) and hBD2(Peprotech), 33 natural compounds (20 ug/ml) are examined for theirability to induce LL-37 and hBD2 from OBA9 cells. Although thesensitivity of the hBD2 ELISA is higher than that of LL-37 ELISA (10pg/ml vs 1 ng/ml, respectively), the induction of LL-37 is moreprominent than hBD2 (FIG. 2 A&B, LL-37; C&D, hBD2). Trihydroxybenzoatederivatives and control mitogenic agents, PMA and Poly IC, inducedLL-37, whereas trihydroxybenzoate derivatives, PMA and Poly IC haveminimal induction activities. Bactericidal effects of LL-37 and hBD2are, in general, potent. Therefore, although hBD2 induction wassignificantly elevated compared to the non-stimulated control, theconcentration of expressed hBD2 appeared insufficient to kill bacteria.

The trihydroxybenzoate derivatives of the Invention and rosemarinicacid, courmarin, catechin gallate, epicatechin gallate,epigallocatechin, and tannic acid are tested to induce LL-37 from OBA9cells in the absence of EGCG. Rosemarinic acid and courmarin, which lack3,4,5-trihydroxy benzoate, showed little or no induction of LL-37expression by OBA9 cells. Therefore, gallic acid appears to be morepotent than 3,4,-dihydroxy benzoate in the induction of LL-37 expressionby cultured gingival epithelial cells.

Culture Supernatant of EGCG-Treated Human Gingival Epithelial Cells.

The culture supernatant isolate from OBA9 is treated with 1) EGCG, 2)catechin, 3) 2,4-dihydroxy benzoic acid, and 4) 3,4,5-tri-hydroxybenzoic acid (20 ug/ml, respectively) and examined for theirbactericidal effects on Actinobacillus actinomycetemcomitans Y4 (Aa Y4).The culture supernatant of EGCG- and 3,4,5-tri-hydroxy benzoicacid-treated OBA9 cells show more bactericidal effects than controlnon-treated OBA9 supernatant or supernatant of OBA9 treated withcatechin or with 2,4-dihydroxy benzoic acid. Therefore,3,4,5-tri-hydroxy benzoic acid and EGCG which contains gallic acid bothappear to have higher antimicrobial effects than the compounds which donot have the structural isomers of gallic acid

In Vivo Induction of LL-37 mRNA and Protein Expression by Human CheekEpithelial Cells.

Cheek epithelium is utilized for monitoring LL-37 mRNA and proteinexpression to determine if Trihydroxybenzoate Derivatives of theInvention can induce LL-37 in vivo. Total RNA is extracted from cheekepithelial cells after application of mouthwash with EGCG (40 ug/ml),and LL-37 mRNA expression by the cheek epithelial cells is determined byRT-PCR. mRNA for LL-37 is up-regulated after only 5 minutes fromapplication of the mouthwash with EGCG, whereas the internal controlb-actin is expressed constitutively. The isolated cheek cells are alsosubjected to Western blot analysis for protein expression of LL-37 bycheek epithelium using anti-LL-37 monoclonal antibody. The expression ofboth the proform (18 kD, also termed as hCAP18) and secreted form (4.5kD) of LL-37 (12) in cheek epithelial cells is induced byTrihydroxybenzoate Derivatives of the Invention (e.g., EGCG (95% SIGMA),or by EGCG (90% Teavigo)). The immunohistochemical analysis for LL-37expression stained with anti-LL-37 monoclonal antibody also demonstratethe increased expression of LL-37 in the cheek epithelial cells after exvivo stimulation.

Tri-Hydroxy Benzoic Acid.

In order to investigate the structure-function relationship underlyingEGCG-mediated induction of LL-37 from human gingival epithelial cellline OBA9, different isomers of benzoic acids are examined for theireffects on the LL-37 expression by OBA9 cells. Each isomer of benzoicacids is coupled to NBS-activated Sepharose gel (Pierce) using across-linker, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC,Pierce). The isomers of benzoic acids include 1) 2,6-di-hydroxy benzoicacid, 2) 2,3,4-tri-hydroxy benzoic acids, 3) 3,4,5-tri-hydroxy benzoicacids, and 4) 2,4,6-tri-hydroxy benzoic acids. Only 2,3,4-tri-hydroxybenzoic acids and 3,4,5-tri-hydroxy benzoic acids, but not2,6-di-hydroxy benzoic acids or 2,4,6-tri-hydroxy benzoic acids, showthe induction of LL-37 expression by OBA9 cells.

Example 2 Binding to DDX5

In order to confirm if LL-37 induction in epithelial cells by EGCG isdependent on the activation of cell ligand (either cytoplasm or cellsurface), or previously known anti-oxidant effects by EGCG,3,4,5-tri-hydroxy benzoic acid-coupled beads and control 2,6-di-hydroxybenzoic acid-coupled beads are utilized to pull down the possible ligandfor 3,4,5-tri-hydroxy benzoic acid. After incubation of each bead withcell lysate of OBA9, the beads are washed, and the cellular materialsthat bind to the beads are separated in SDS-PAGE. At least 3 bands (MW35 kD, 70 kD and 90 kD), which are distinct to 3,4,5-tri-hydroxy benzoicacid, are detected, as compared to the other beads (control,2,6-di-hydroxy benzoic acid and control plain beads). The 3 bands (35kD, 70 kD and 90 kD) are cut out from SDS-PAGE gel and submitted forMass-Spectrometry analysis service at Tufts University Core Facility.While the 35 kD and 90 kD bands do not contain any distinct protein to3,4,5-tri-hydroxy benzoic acid, the 70 kD band appears to contain DEADbox polypeptide 5 (DDX5)(FIG. 2). This result indicates that3,4,5-tri-hydroxy benzoic acid binds to DDX5 expressed in gingivalepithelial cells with high affinity, and provides strong evidence thatTHBC-mediated LL-37 expression in epithelial cells does result from theligation of DDX5 by THBC present in EGCG.

Direct targeting of DDX5 by synthetic chemicals different from THBC, orby protein agonists, should result in the development of additionalforms of regimens that increase the LL-37 expression by epithelialcells.

Our hypothesis that THBC induction of LL-37 expression is mediated viabinding to DDX5 is further supported by the finding that inhibition ofDDX5 expression in gingival epithelial cells (OBA9) reducesEGCG-mediated LL-37 expression. Confluent OBA9 cells in a tissue cultureplate are treated with or without DDX5-specific siRNA sequences (threedifferent siRNA sequences specific to DDX5 are examined). After 24 h and48 h incubation, total RNA and proteins are extracted, respectively,from the OBA9 cells. The results of RT-PCR for DDX5 mRNA and internalcontrol of β-actin mRNA and the results of Western blot for DDX5 proteindemonstrate that all three different DDX5 siRNA sequences remarkablysuppress the expression of DDX5 at both mRNA and protein levels. TheOBA9 cells pre-incubated with non-target siRNA and DDX5-siRNA for 48hours are restimulated with EGCG (40 ng/ml) for 30 min, and mRNAexpression for LL-37 and 13-actin was detected by RT-PCR. Veryimportantly, LL-37 expression induced in OBA9 cells by EGCG stimulationis remarkably suppressed by pretreatment of OBA9 cells with DDX5-siRNA.Effects of DDX5-siRNA treatment on EGCG-mediated LL-37 proteinexpression by OBA9 cells is measured by ELISA. The EGCG-mediatedinduction of LL-37 production is detected as early as 30 min until 24hours after EGCG stimulation in the OBA9 cells which do not receivepretreatment (medium alone) or which are treated with control non-targetsiRNA sequence. However, inhibition of DDX5 expression using DDX5-siRNAin OBA9 cells significantly suppresses the EGCG-mediated LL-37production by OBA9 cells for all incubation periods examined (from 30min to 24 h). These results indicate that ligation of DDX5 with3,4,5-THBC present in EGCG can induce LL-37 expression from gingivalepithelial cells.

Example 3 Formulations

In one embodiment, the composition of the invention may be preparedusing the following ingredients:

TABLE 1 MATERIAL WEIGHT % Polyphenol extract-I 0.200 Trihydroxybenzoatederivatives 0.050 Carboxymethyl cellulose 1.100 Sodium Saccharin 0.200Sodium Monofluorophosphate 1.100 Tetrasodium pyrophosphate 0.500Phosphoric acid 85% 0.600 Dicalcium phosphate dihydrate 46.500 Sodiumlauryl sulfate 35% 4.071 Flavor 0.950 Glycerin 22.000 Deionized WaterBalance TOTAL 100.000wherein polyphenol extract-1 comprises between 0.06-0.6% gallic acid.

In another embodiment, the composition of the invention may be preparedusing the following ingredients:

TABLE 1 MATERIAL WEIGHT % Polyphenol extract-2 0.200 Trihydroxybenzoatederivatives 0.050 Carboxymethyl cellulose 1.100 Sodium Saccharin 0.200Sodium Monofluorophosphate 1.100 Tetrasodium pyrophosphate 0.500Phosphoric acid 85% 0.600 Dicalcium phosphate dihydrate 46.500 Sodiumlauryl sulfate 35% 4.071 Flavor 0.950 Glycerin 22.000 Deionized WaterBalance TOTAL 100.000wherein polyphenol extract-2 comprises less than 0.04% of catechins andepicatechins. In yet another embodiment, the composition of theinvention comprises the ingredients disclosed in Table 3, whereinpolyphenol extract-2 contains less than less than 0.04% of catechins andepicatechins and less than 0.005% of GCG (Polyphenol extract-3).

In a certain embodiment, the compositions of the invention is asfollows:

Toothpaste-Formulation A MATERIAL WEIGHT % Polyphenol extract-I 0.200Trihydroxybenzoate derivatives 0.050 Dibasic calcium phosphate 42.000Glycerine 18.000 Sodium lauryl sulfate 1.200 Saccharin sodium 0.090Flavoring agent 1.000 Water Balance TOTAL 100.000

Toothpaste-Formulation B MATERIAL WEIGHT % Polyphenol extract-2 orextract-3 0.200 Trihydroxybenzoate derivatives 0.050 Dibasic calciumphosphate 42.000 Glycerine 18.000 Sodium lauryl sulfate 1.200 Saccharinsodium 0.090 Flavoring agent 1.000 Water Balance TOTAL 100.000

Toothpaste-Formulation C MATERIAL WEIGHT % Polyphenol extract 0.001Trihydroxybenzoate derivatives 0.250 Dibasic calcium phosphate 42.000Glycerine 18.000 Sodium lauryl sulfate 1.200 Saccharin sodium 0.090Flavoring agent 1.000 Water Balance TOTAL 100.000

Toothpaste-Formulation D MATERIAL WEIGHT % Polyphenol extract-I 0.100Trihydroxybenzoate derivatives 0.050 Dibasic calcium phosphate 42.000Glycerine 18.000 Sodium lauryl sulfate 1.200 Saccharin sodium 0.090Flavoring agent 1.000 Water Balance TOTAL 100.000

Mouth wash-Formulation A MATERIAL WEIGHT % Polyphenol extract-I 0.200Trihydroxybenzoate derivatives 0.050 Sodium lauryl sulfate 0.800Glycerine 7.000 Sorbitol 5.000 Ethyl alcohol 15.000 !-Menthol 0.050Flavoring agent 0.040 Saccharin sodium 0.100 Water Balance TOTAL 100.000

Mouth wash-Formulation B MATERIAL WEIGHT % Polyphenol extract-2 orextract-3 0.200 Trihydroxybenzoate derivatives 0.050 Sodium laurylsulfate 0.800 Glycerine 7.000 Sorbitol 5.000 Ethyl alcohol 15.000!-Menthol 0.050 Flavoring agent 0.040 Saccharin sodium 0.100 WaterBalance TOTAL 100.000

Mouth wash-Formulation C MATERIAL WEIGHT % Polyphenol extract-I 0.001Trihydroxybenzoate derivatives 0.250 Sodium lauryl sulfate 0.800Glycerine 7.000 Sorbitol 5.000 Ethyl alcohol 15.000 !-Menthol 0.050Flavoring agent 0.040 Saccharin sodium 0.100 Water Balance TOTAL 100.000

Mouth wash-Formulation D MATERIAL WEIGHT % Polyphenol extract-I 0.100Trihydroxybenzoate derivatives 0.050 Sodium lauryl sulfate 0.800Glycerine 7.000 Sorbitol 5.000 Ethyl alcohol 15.000 !-Menthol 0.050Flavoring agent 0.040 Saccharin sodium 0.100 Water Balance TOTAL 100.000

The examples described herewith are intended to demonstrate illustrativeembodiments within the scope of the present invention. The examples aregiven solely for illustration and are not to be construed as limitationsof this invention as many variations are possible without departing fromthe spirit and scope thereof. Various modifications of the invention inaddition to those shown and described herein should be apparent to thoseskilled in the art and are intended to fall within the appended claims.

Example 4 Method of Treatment

The effectiveness of the compositions of the present invention forimprovement of oral health can be tested by measuring plaqueaccumulation over a period of time. The experiments can be conducted asfollows:

A study can be carried to test the effect of a new formulation of atoothpaste containing trihydroxybenzoate derivatives on dental plaqueformation. Slurries of the toothpastes being tested are prepared andused as rinses, with subjects abstaining from mechanical plaque controlfor 4 days. The slurry method is used because mechanical plaque controlalone, performed by a skilled person, would reduce dental plaqueformation, with or without the use of a toothpaste or antibacterialagent. A mouthwash formulation containing 0.5 wt. % of Oolong teaextract is chosen as a positive control.

Subject Volunteers participating in the study are non-smokers, in goodgeneral health and included both women and men between 20 and 55 yearsof age. They do not use any mouthwash or toothpaste containingTrihydroxybenzoate Derivatives of the Invention as part of their routineoral hygiene practices. The subjects may be chosen by the large amountsof plaque formed in the past. The trial is conducted over a period ofseveral weeks, with the subjects attending a dental clinic on themorning of the first day for a prophylaxis. The subjects are thenprovided with one of three blinded samples to be used as a rinse on theevening of day 1; twice daily, morning and evening, for days 2, 3, 4;and once in the morning of day 5. Mechanical plaque control is notallowed during this period. On the morning of day 5, the subjects againattended the clinic, the plaque on their teeth is disclosed by theapplication of a dye, and a score in the range of 0-5 (where 0 was noplaque, 5 was maximum plaque) for individual teeth is determined. Theteeth are then photographed and given a prophylaxis. The subjects arethen allowed to resume their habitual plaque control for a week. On thefirst day of the third week, the whole process is repeated, with thesubject receiving a different preparation to trial, and so on until allfour samples have been tested. One of the randomized samples is a greentea extract mouthwash used as a control. At no time does the subject orthe examiner know which preparation was being used. The threepreparations are randomly distributed amongst the subjects in such amanner that all subjects use all four preparations but in varying order.The key is held in a secure location until the completion of the trial.

The samples that may be used are: (1) toothpaste of the presentinvention, mixed in a 15 ml slurry; (2) formulation containing 0.5 wt. %of Oolong tea extract is chosen as a positive control; and (3) 3 g ofPlacebo toothpaste, mixed in a 15 ml slurry. The teeth are examinedafter 4 days, and a dye was applied to highlight the plaque.

The experiment will show that the control mouthwash provides theanticipated level of plaque protection while preparations (1) and (2)above both perform significantly better than the placebo.

Second Trial

This trial focuses on microbiology, particularly changes/shifts in theflora. A mouthwash with the trihydroxybenzoate derivatives isadministered 2× per day with 15 ml rinse solution, 1 minute rinse.Salivary microflora are analyzed post use at 0, 30 minutes, 60 minutesand 3 hours. LL-37 levels in whole saliva are measured at various timepoints. The panelists are instructed not to consume any food betweenmeasurements. The concentrations of LL-37 in saliva and GCF aremeasured. Checker board method is used for enumerating bacteria insaliva. A blood agar plate is used to measure total viable bacteriacount in the saliva. This provides a measure of attachment loss. Thefollowing results are observed: a significant increase of LL-37 insaliva and GCF, a significant reduction of total viable bacterialaccount vs placebo, and a shift of the oral bacterial microflora.

Example 5 The effect of DDX5 on LL-37

Since DDX5 was found to be a possible receptor for 2,3,4- and3,4,5-trihydroxybenzoic acids, and EGCG, based on the pull down assay,the engagement of DDXS in the EGCG mediated up-regulation of LL-37expression in human gingival epithelial cells was examined by functionalgenomic approaches.

1) Effects of DDX5-Specific RNA Interference (RNAi) on the EGCG-InducedLL-37 Expression in Human Gingival Epithelial Cells:

1-A) Efficacy of DDX5-Specific siRNA Sequences on the Suppression ofDDX5 mRNA; Evaluation of RNAi Assay System (FIG. 3):

Confluent OBA-9 cultured in 24-well plate was reacted with threedifferent sequences of siRNA for DDX5 which were designed and producedby a commercial service (Ambion, Austin, Tex.). As a control, scrambledsiRNA sequence (Ambion) was used. Each siRNA was applied to the OBA9cells in the mixture with liposome-based transfection reagent(DhermaFECT No.2, Thermo Scientific Dharmacon, Chicago Ill.). To confirmthe efficiency of each siRNA to suppress DDX5 mRNA, total RNA wasisolated from the cells after incubation with for siRNA for 24 h,followed by the RT-PCR for DDX5 mRNA. In order to examine the expressionlevel of DDX5 protein, OBA-9 reacted with siRNA for 48 h was subjectedto the Western-blot analysis. As shown in FIG. 3, both DDX5 mRNA as wellas DDX5 protein was constitutively expressed in OBA9 cells. All threeexamined siRNA sequences for DDX5 mRNA showed remarkable suppressioneffects on the both expressions of DDX5 mRNA (RT-PCR) as well as DDX5protein (Western blot). The expression of internal control 13-actin mRNAwas not affected by the treatment with any siRNA. The control scrambledsequence of control siRNA (non-target siRNA) did not affect theexpression of DDX5 mRNA as well as protein. These results demonstratedthat mRNA DDX5-specific RNAi can suppress the expression of DDX5protein, which is the receptor for 2,3,4-trihydroxybenzoic acid, andEGCG.

1-B) The Effect of DDX5-Specific RNAi on the EGCG-Induced LL37 mRNAExpression (FIG. 4):

After treatment of OBA9 cells with control non-target siRNA orDDX5-siRNA (#A) for 48 hours, the OBA9 cells were incubated with orwithout EGCG (40 ng/ml) for 30 minutes. The total RNA isolated from theOBA9 cells was subjected to RT-PCR for LL37 mRNA as well as internalcontrol β-actin mRNA. As shown in FIG. 4, the LL37 mRNA expressioninduced by EGCG was inhibited by the pretreatment of OBA9 cells withDDX5-siRNA sequence #A and #B but not with control non-target siRNA,indicating that DDX5-RNAi suppress the LL-37 mRNA expression induced byEGCG.

1-C) the Effect of DDX5-Specific RNAi on the EGCG-Induced LL37 ProteinExpression (FIG. 5):

The OBA9 cells which were pre-treated with control non-target siRNA orDDX5-siRNA (#A) for 48 hours, as shown in 1-B, were incubated with EGCG(40 ng/ml) for different periods from 30 min to 24 hours. Thesupernatant harvested from the OBA9 cell culture was subjected to ELISAfor LL37 protein. As shown in FIG. 5, the LL37 expression was observedas early as 30 minutes after the EGCG stimulation and the expressionlevel of LL37 increased in a time dependent manner. While controlnon-target siRNA did not affect the LL-37 expression induced by EGCG,DDX5-siRNA significantly inhibited the LL-37 expression induced by EGCGtreatment for the periods from 30 min to 24 hours (*, P<0.05, Student ttest). This result indicated that DDX5 is involved in the EGCG-mediatedLL-37 protein expression in OBA9 cells.

2) Effects of Over Expression of DDX5 on the EGCG-Induced LL-37Expression in Human Gingival Epithelial Cell Line, OBA9:

If DDX5 play a role as a receptor for EGCG in which action to induceLL-37 expression, over expression of DDX5 in human gingival epithelialcells (HGEC) using a molecular genetic technology should up-regulate theLL-37 expression by the EGCG-stimulated HGEC.

2-A) Development of Vector-Mediated DDX5 Over-Expression in OBA9 Cells(FIG. 6):

Open reading frame (ORF) of human DDX5 cDNA isolated from OBA9 cells wasamplified and cloned into pGEM T-easy vector (Promega, Madison Wis.). Inorder to construct DDX5 over expression vector, the fragment of DDX5cDNA was ligated into piRESneo3 vector (Promega). The DDX5 overexpression vector was designated as pK0203, while the control emptyvector was termed as pK0108.

OBA-9 was cultured as the same condition described previously. Eachvector was transfected to OBA9 cells using Lipofectamine LTX(Invitrogen).

To confirm if DDX5 over expression vector (pK0203) can increase theexpression of DDX5 mRNA as well as DDX5 protein, OBA9 cells received theexpression vector were cultures for 24 h or 48 h, and subjected toRT-PCR and Western-blot analyses, respectively. As shown in FIG. 6, DDXSover-expression vector (pK0203) up-regulated the expression of DDX5 mRNAas well as DDX5 protein in a dose dependent manner. Contrast to pK0203,control empty vector (pK0108) did not increase the expression of DDX5mRNA or DDX5 protein.

2-B) the Effect of DDX5 Over Expression on the Induction of LL37Mediated by EGCG (FIG. 7):

The OBA9 cells transfected with either DDX5 over-expression vector(pK0203) or control empty vector (pKO108) were cultured for 48 h.Thereafter, the OBA9 cells were incubated with or without EGCG (40ng/ml) for 2 hours, and the culture supernatant was subjected to ELISAfor the detection of LL-37.

As shown in FIG. 7, EGCG induced the LL-37 expression in all threeconditions of OBA9 treatment, i.e. 1) control OBA9 received no vector,2) OBA9 cells transfected with control empty vector (pK0108), and 3)OBA9 cells transfected with DDX5 over-expression vector (pK0203).

Most importantly, LL-37 expression induced by EGCG was significantlyhigher in the OBA9 cells transfected with DDX5 over-expression vectorthan 1) control OBA9 received no vector or 2) OBA9 cells transfectedwith control empty vector (pKO108) (*, P<0.05, Student t test).Therefore, this result indicated that increased amount of DDX5 canpromote the EGCG-mediated LL-37 protein expression in OBA9 cells.

Based on the results obtained from loss-of-function assay using RNAitechnology and gain-of-function assay using vector-based overexpression, DDX5 functions as a receptor for EGCG, as well as 2,3,4- and3,4,5-trihydroxybenzoic acids.

The relevant teachings of all the references, patents and/or patentapplications cited herein are incorporated herein by reference in theirentirety.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

The entire teachings of the following application are incorporatedherein by reference: U.S. application Ser. No. (Attorney docket no:0024.0012-001) filed on Apr. 16, 2010, entitled “New Methods of MakingAn Antibody and Compositions Thereof” by Toshihisa Kawai, et al.

1-6. (canceled)
 7. A method of treating or preventing an oral cavitydisease or condition in an individual, the method comprisingadministering an trihydroxybenzoate derivatives a therapeuticallyeffective amount of one or more compounds having one or moretrihydroxybenzoate moieties to the oral cavity of the individual;wherein the stimulation of the production of antimicrobial peptidesoccurs to thereby treat or prevent the oral cavity disease or conditionin the individual.
 8. The method of claim 7, wherein the oral cavitydisease or condition comprises thrush, gingivitis, periodontitis, oralcandidiasis, salivary diseases associated with bacterial, viral, orfungal infections; Crohn's disease, irritable bowel syndrome, ulcerativecolitis, Helicobacter pylori caused stomach ulcer, and gastrointestinaldiseases associated with mucosal bacteria.
 9. The method of claim 8,wherein stimulation of the production of antimicrobial peptide reducesone or more symptoms associated with the oral cavity disease orcondition. 10-16. (canceled)
 17. A method of treating or preventing anoral cavity disease or condition in an individual, the method comprisingadministering a therapeutically effective amount of one or morecompounds having one or more trihydroxybenzoate moieties to the oralcavity of the individual; whereby oral bacteria are reduced or inhibitedto thereby treat or prevent the oral cavity disease or condition in theindividual.
 18. The method according to claim 17, wherein a level ofantimicrobial LL-37 peptides in the oral cavity is increased, ascompared to a level of antimicrobial LL-37 peptides prior toadministration of the oral care composition. 19-42. (canceled)